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Are lower magnification objectives brighter?

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0000001ed7f52e4a-dmarc-request

Are lower magnification objectives brighter?

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Dear all,
Are lower magnification objectives brighter than higher magnification ones when they have the same NA, e.g. a 40x NA 1.4 objective compared to 63x NA 1.4? I mean for confocal microscopy.

Confocal.nl stated this is a recent webinar and on their website:
“A lower magnification allows for a larger field of view and brighter images, since light intensity is inversely proportional to the magnification squared”https://www.confocal.nl/#rcm2

I would think that this is caused by less light going through the smaller back focal aperture when the illumination is held constant? Most of the light is clipped as explained in fig 1 of https://www.nature.com/articles/s41596-020-0313-9
So, the microscope manufacturer could adjust the illumination beam path and laser powers to best suit the objective?Or are lower magnification objectives really brighter?

The field of view will obviously be larger for the 40x objective, but I am more interested to understand the claimed benefit in brightness.

best wishes

Andreas

Michael Giacomelli-2

Re: [EXT] Are lower magnification objectives brighter?

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Hi Andreas,

If you divide the same amount of light across a more magnified PSF, then
the PSF covers more pixels and so each pixel gets fewer photons. However,
in this case you would also be more densely sampled, and you could
digitally downsample the image, which would have the effect of putting the
same number photons into fewer pixels. If dark and read noise are low,
this would effectively give you the same image as you would have gotten
using a lower magnification to begin with.

Mike

On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
[hidden email]> wrote:

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> and include the link in your posting.
> *****
>
> Dear all,
> Are lower magnification objectives brighter than higher magnification ones
> when they have the same NA, e.g. a 40x NA 1.4 objective compared to 63x NA
> 1.4? I mean for confocal microscopy.
>
> Confocal.nl stated this is a recent webinar and on their website:
> “A lower magnification allows for a larger field of view and brighter
> images, since light intensity is inversely proportional to the
> magnification squared”
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
>
> I would think that this is caused by less light going through the smaller
> back focal aperture when the illumination is held constant? Most of the
> light is clipped as explained in fig 1 of
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63nsf-C6L2XiGYA&e=
> So, the microscope manufacturer could adjust the illumination beam path
> and laser powers to best suit the objective?Or are lower magnification
> objectives really brighter?
>
> The field of view will obviously be larger for the 40x objective, but I am
> more interested to understand the claimed benefit in brightness.
>
> best wishes
>
> Andreas
>

Christoph Ruediger Bauer

Re: Are lower magnification objectives brighter?

In reply to this post by 0000001ed7f52e4a-dmarc-request

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Hi,

I teach my students that light gathering power of an objective depends on the NA raised to the power of four divided by its magnification squared. I think the concept comes from Shinya Inoué probably in his book "video microscopy" if I remember correctly.
Not sure if the confocal setup is changing this principle as it may not always make full use of the NA (for example when using a beam expander).
Best regards,
Christoph

BIOIMAGING CENTER
University of Geneva - Science II
Room 245
30, Quai Ernest Ansermet
CH - 1211 Genève 4

Dr. Christoph R. Bauer
Managing Director
Tel.: + 41 22 3796632
Fax: + 41 22 3796868
email: [hidden email]
website: http://bioimaging.unige.ch/

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Andreas Bruckbauer
Sent: 22 March 2021 17:28
To: [hidden email]
Subject: Are lower magnification objectives brighter?

*****
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*****

Dear all,
Are lower magnification objectives brighter than higher magnification ones when they have the same NA, e.g. a 40x NA 1.4 objective compared to 63x NA 1.4? I mean for confocal microscopy.

Confocal.nl stated this is a recent webinar and on their website:
“A lower magnification allows for a larger field of view and brighter images, since light intensity is inversely proportional to the magnification squared”https://www.confocal.nl/#rcm2

I would think that this is caused by less light going through the smaller back focal aperture when the illumination is held constant? Most of the light is clipped as explained in fig 1 of https://www.nature.com/articles/s41596-020-0313-9
So, the microscope manufacturer could adjust the illumination beam path and laser powers to best suit the objective?Or are lower magnification objectives really brighter?

The field of view will obviously be larger for the 40x objective, but I am more interested to understand the claimed benefit in brightness.

best wishes

Andreas

lechristophe

Re: Are lower magnification objectives brighter?

In reply to this post by 0000001ed7f52e4a-dmarc-request

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*****

Hi Andreas,

I've always understood this in relationship to a constant detector with a
given pixel size (like a camera): lower magnification spreads the same
signal over a smaller number of pixels, resulting in higher intensity for
the pixels that contain the signal. This is more tricky with point-scanning
microscopes.

Christophe

On Mon, 22 Mar 2021 at 18:02, Andreas Bruckbauer <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
> Are lower magnification objectives brighter than higher magnification ones
> when they have the same NA, e.g. a 40x NA 1.4 objective compared to 63x NA
> 1.4? I mean for confocal microscopy.
>
> Confocal.nl stated this is a recent webinar and on their website:
> “A lower magnification allows for a larger field of view and brighter
> images, since light intensity is inversely proportional to the
> magnification squared”https://www.confocal.nl/#rcm2
>
> I would think that this is caused by less light going through the smaller
> back focal aperture when the illumination is held constant? Most of the
> light is clipped as explained in fig 1 of
> https://www.nature.com/articles/s41596-020-0313-9
> So, the microscope manufacturer could adjust the illumination beam path
> and laser powers to best suit the objective?Or are lower magnification
> objectives really brighter?
>
> The field of view will obviously be larger for the 40x objective, but I am
> more interested to understand the claimed benefit in brightness.
>
> best wishes
>
> Andreas
>

Jonkman, James

Re: Are lower magnification objectives brighter?

In reply to this post by 0000001ed7f52e4a-dmarc-request

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Hi, Andreas. It bothered me for many years that people still claimed that a CLSM gives you brighter images when you use a lower magnification objective (for the same NA). Physically, it didn't make sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal. If you consider the focused spot on a CLSM, the size of the PSF depends only on the NA of the objective and not it's magnification, so the illumination will be identical for a 40x and a 63x objective with the same NA (assuming that you overfill the back aperture in both cases to take full advantage of the NA of the lens). Now consider the detection: again, only the NA determines how much light you will collect by the lens. So it wouldn’t make any sense for a CLSM to give you a "brighter" image with a lower mag lens when both lenses have the same NA.

But wait! When you look into the binocular it looks brighter with the 40x lens. AND, if you keep all of the same settings (laser power percentage and detector gain) you get a brighter image with the 40x objective. So what's going on? My relatively new Thorlabs power meter (PM400 console with S170C sensor) is compatible with oil immersion and the difference in brightness with the 40x objective is 100% accounted for by the change in laser power when you switch between these objectives. The change in laser power is due to the smaller back aperture of the 63x objective. In other words, when you switch from the 40x to the 63x objective, the edges of the laser beam are blocked by the smaller aperture of the 63x lens, so less excitation reaches the sample. If you adjust the % laser power slider so that both the 40x and 63x objectives are reading the same illumination intensity, then you get the exact same image with both lenses.

As you mentioned, I tried to explain this in our Nat Prot paper in Supplementary Figure 1 and I included some of the data there (free download for the Supp Figs - for the full paper if anyone needs it I'm happy to email it to them).
https://www.nature.com/articles/s41596-020-0313-9

So why is this so broadly misunderstood (I have heard it many, many times!)? When we read the classic textbooks on the brightness of a microscope image, these were originally written with respect to transmitted-light brightfield microscopy: it's not obvious that they should apply to confocal microscopy or even to widefield fluorescence microscopy. On the Microscopy Primer website (https://www.microscopyu.com/microscopy-basics/image-brightness ), for example, they start with the typical statement that the Image Brightness is proportional to (NA/M)^2. They go on to mention that for fluorescence the Image Brightness should be lambda NA^4/ M^2. However, they fail to mention that the reason for the Mag being in the denominator of the equation is because the size of the back aperature depends on Mag in this way. So even for a widefield fluorescence microscope, the increase in brightness is caused by increased illumination on the sample, not increased detection efficiency, which is not very helpful in this era of over-powered fluorescence lamps.

If the confocal manufacturers would specify their laser powers in real-world units instead of %_of_maximum, when you switch lenses you would immediately see that that for a given excitation power density (in W/cm^2) you get the same intensity image for 2 lenses with the same NA, regardless of the mag of the lens.

Cheers,
James

-----------------------------------------------
James Jonkman, Staff Scientist
Advanced Optical Microscopy Facility (AOMF)
and Wright Cell Imaging Facility (WCIF)
University Health Network
MaRS, PMCRT tower, 101 College St., Room 15-305
Toronto, ON, CANADA M5G 1L7
[hidden email] Tel: 416-581-8593
www.aomf.ca

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Giacomelli
Sent: Monday, March 22, 2021 1:10 PM
To: [hidden email]
Subject: [External] Re: [EXT] Are lower magnification objectives brighter?

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Hi Andreas,

If you divide the same amount of light across a more magnified PSF, then the PSF covers more pixels and so each pixel gets fewer photons. However, in this case you would also be more densely sampled, and you could digitally downsample the image, which would have the effect of putting the same number photons into fewer pixels. If dark and read noise are low, this would effectively give you the same image as you would have gotten using a lower magnification to begin with.

Mike

On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer < [hidden email]> wrote:

> *****
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> and include the link in your posting.
> *****
>
> Dear all,
> Are lower magnification objectives brighter than higher magnification
> ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> to 63x NA 1.4? I mean for confocal microscopy.
>
> Confocal.nl stated this is a recent webinar and on their website:
> “A lower magnification allows for a larger field of view and brighter
> images, since light intensity is inversely proportional to the
> magnification squared”
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
>
> I would think that this is caused by less light going through the
> smaller back focal aperture when the illumination is held constant?
> Most of the light is clipped as explained in fig 1 of
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> illumination beam path and laser powers to best suit the objective?Or
> are lower magnification objectives really brighter?
>
> The field of view will obviously be larger for the 40x objective, but
> I am more interested to understand the claimed benefit in brightness.
>
> best wishes
>
> Andreas
>

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Tim Feinstein

Re: Are lower magnification objectives brighter?

In reply to this post by lechristophe

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You have to take the NA into account. I have not looked up the physics, but Christoph Ruediger's heuristic sounds about right.

You have to take coatings and lens elements for correcting chroma, flat field, etc into account as well, as each element & coating will necessitate some light loss. I recall the brightest objective in one scope maker's product line (at the time that I asked) was a 40x 1.3 with modest chroma correction that hit a sweet spot between magnification, NA, and the number of lens elements & coatings.

That makes me wonder whether microscopy has anything like the famous 50mm/1.8 lenses for photography. That length & aperture hit an engineering 'sweet spot' that let you get near-perfect optical quality with a minimum number of all-spherical lens elements. Every manual SLR camera used to come with one because the makers could all produce amazing ones in a compact size and basically for free. If such a thing exists in microscopy I haven't heard about it.

All the best,

T

Timothy Feinstein, Ph.D.

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Christophe Leterrier
Sent: Monday, March 22, 2021 1:21 PM
To: [hidden email]
Subject: Re: Are lower magnification objectives brighter?

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Hi Andreas,

I've always understood this in relationship to a constant detector with a given pixel size (like a camera): lower magnification spreads the same signal over a smaller number of pixels, resulting in higher intensity for the pixels that contain the signal. This is more tricky with point-scanning microscopes.

Christophe

On Mon, 22 Mar 2021 at 18:02, Andreas Bruckbauer < [hidden email]> wrote:

> *****
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> *****
>
> Dear all,
> Are lower magnification objectives brighter than higher magnification
> ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> to 63x NA 1.4? I mean for confocal microscopy.
>
> Confocal.nl stated this is a recent webinar and on their website:
> "A lower magnification allows for a larger field of view and brighter
> images, since light intensity is inversely proportional to the
> magnification
> squared"<a href="https://nam12.safelinks.protection.outlook.com/?url=https%3A%2">https://nam12.safelinks.protection.outlook.com/?url=https%3A%2
> F%2Fwww.confocal.nl%2F%23rcm2&data=04%7C01%7Ctnf8%40PITT.EDU%7Cf2c
> 11405023a4bd8240908d8ed575e19%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7
> C0%7C637520306784421783%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLC
> JQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=eeJv0l7y
> 2Th7bBYqEwJtUkDMrs4wNy%2F10%2F8RsfWGrfE%3D&reserved=0
>
> I would think that this is caused by less light going through the
> smaller back focal aperture when the illumination is held constant?
> Most of the light is clipped as explained in fig 1 of
> https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Ctnf8%40PI
> TT.EDU%7Cf2c11405023a4bd8240908d8ed575e19%7C9ef9f489e0a04eeb87cc3a5261
> 12fd0d%7C1%7C0%7C637520306784421783%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC
> 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sd
> ata=NMagM%2FIuo%2BVswcZuCnx%2B5GIO92Mv6m%2BGxoA9AOES%2BoM%3D&reser
> ved=0 So, the microscope manufacturer could adjust the illumination
> beam path and laser powers to best suit the objective?Or are lower
> magnification objectives really brighter?
>
> The field of view will obviously be larger for the 40x objective, but
> I am more interested to understand the claimed benefit in brightness.
>
> best wishes
>
> Andreas
>

Craig Brideau

Re: Are lower magnification objectives brighter?

In reply to this post by Jonkman, James

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Thanks for the great answer James! For additional information, here's some
power readings from one of our confocals at various wavelengths. As you can
see between the 20x and 60x there is considerable variability by laser
color as well as by magnification. Units are in microwatts.
Intensity measured (in micro watt) using 20X (air) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 78 233 400 555
457 4 7 10 11
476 10 19 27 35
488 67 133 195 246
514 27 53 78 98
561 195 380 555 700
638 no data no data no data no data
Intensity measured (in micro watt) using 60X (oil) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 14 28 63 77
457 0 0 2.3 3
476 3 6 8 10
488 18 35 51 66
514 9 17 26 32
561 73 141 203 261
638 no data no data no data no data
0 : value under detection level
Craig

On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, Andreas. It bothered me for many years that people still claimed that
> a CLSM gives you brighter images when you use a lower magnification
> objective (for the same NA). Physically, it didn't make sense to me. I
> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> If you consider the focused spot on a CLSM, the size of the PSF depends
> only on the NA of the objective and not it's magnification, so the
> illumination will be identical for a 40x and a 63x objective with the same
> NA (assuming that you overfill the back aperture in both cases to take full
> advantage of the NA of the lens). Now consider the detection: again, only
> the NA determines how much light you will collect by the lens. So it
> wouldn’t make any sense for a CLSM to give you a "brighter" image with a
> lower mag lens when both lenses have the same NA.
>
> But wait! When you look into the binocular it looks brighter with the 40x
> lens. AND, if you keep all of the same settings (laser power percentage
> and detector gain) you get a brighter image with the 40x objective. So
> what's going on? My relatively new Thorlabs power meter (PM400 console
> with S170C sensor) is compatible with oil immersion and the difference in
> brightness with the 40x objective is 100% accounted for by the change in
> laser power when you switch between these objectives. The change in laser
> power is due to the smaller back aperture of the 63x objective. In other
> words, when you switch from the 40x to the 63x objective, the edges of the
> laser beam are blocked by the smaller aperture of the 63x lens, so less
> excitation reaches the sample. If you adjust the % laser power slider so
> that both the 40x and 63x objectives are reading the same illumination
> intensity, then you get the exact same image with both lenses.
>
> As you mentioned, I tried to explain this in our Nat Prot paper in
> Supplementary Figure 1 and I included some of the data there (free download
> for the Supp Figs - for the full paper if anyone needs it I'm happy to
> email it to them).
> https://www.nature.com/articles/s41596-020-0313-9
>
> So why is this so broadly misunderstood (I have heard it many, many
> times!)? When we read the classic textbooks on the brightness of a
> microscope image, these were originally written with respect to
> transmitted-light brightfield microscopy: it's not obvious that they should
> apply to confocal microscopy or even to widefield fluorescence microscopy.
> On the Microscopy Primer website (
> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> example, they start with the typical statement that the Image Brightness is
> proportional to (NA/M)^2. They go on to mention that for fluorescence the
> Image Brightness should be lambda NA^4/ M^2. However, they fail to mention
> that the reason for the Mag being in the denominator of the equation is
> because the size of the back aperature depends on Mag in this way. So even
> for a widefield fluorescence microscope, the increase in brightness is
> caused by increased illumination on the sample, not increased detection
> efficiency, which is not very helpful in this era of over-powered
> fluorescence lamps.
>
> If the confocal manufacturers would specify their laser powers in
> real-world units instead of %_of_maximum, when you switch lenses you would
> immediately see that that for a given excitation power density (in W/cm^2)
> you get the same intensity image for 2 lenses with the same NA, regardless
> of the mag of the lens.
>
> Cheers,
> James
>
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [hidden email]
> Subject: [External] Re: [EXT] Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Hi Andreas,
>
> If you divide the same amount of light across a more magnified PSF, then
> the PSF covers more pixels and so each pixel gets fewer photons. However,
> in this case you would also be more densely sampled, and you could
> digitally downsample the image, which would have the effect of putting the
> same number photons into fewer pixels. If dark and read noise are low,
> this would effectively give you the same image as you would have gotten
> using a lower magnification to begin with.
>
> Mike
>
> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> > 65dOAWbgN2OxNnKaw&e=
> > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> > qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > and include the link in your posting.
> > *****
> >
> > Dear all,
> > Are lower magnification objectives brighter than higher magnification
> > ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> > to 63x NA 1.4? I mean for confocal microscopy.
> >
> > Confocal.nl stated this is a recent webinar and on their website:
> > “A lower magnification allows for a larger field of view and brighter
> > images, since light intensity is inversely proportional to the
> > magnification squared”
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >
> > I would think that this is caused by less light going through the
> > smaller back focal aperture when the illumination is held constant?
> > Most of the light is clipped as explained in fig 1 of
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> > nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > illumination beam path and laser powers to best suit the objective?Or
> > are lower magnification objectives really brighter?
> >
> > The field of view will obviously be larger for the 40x objective, but
> > I am more interested to understand the claimed benefit in brightness.
> >
> > best wishes
> >
> > Andreas
> >
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
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> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
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> Please note that we are unable to automatically unsubscribe individuals
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> UHN patients may provide their consent to communicate with UHN about their
> care using email. All electronic communication carries some risk. Please
> visit our website here<
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>

Konstantín Levitskiy

Re: Are lower magnification objectives brighter?

*****
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Hi Craig.
I wonder your drastical decrease power in your Argon laser in 514 line respect to 488 one. We had similar problem in one of the Ar laser we had with warranty and they had to change the laser. The power of 514 nm line has not to be too small than the 488, I think (https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1)
[https://i1.rgstatic.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/links/09e4150f7f8c30a99c000000/largepreview.png]<https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1>
(PDF) Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope<https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1>
PDF | Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and... | Find, read and cite all the research you need on ResearchGate
www.researchgate.net
Regards,
Konstantin

________________________________
De: Confocal Microscopy List <[hidden email]> en nombre de Craig Brideau <[hidden email]>
Enviado: lunes, 22 de marzo de 2021 20:00
Para: [hidden email] <[hidden email]>
Asunto: Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thanks for the great answer James! For additional information, here's some
power readings from one of our confocals at various wavelengths. As you can
see between the 20x and 60x there is considerable variability by laser
color as well as by magnification. Units are in microwatts.
Intensity measured (in micro watt) using 20X (air) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 78 233 400 555
457 4 7 10 11
476 10 19 27 35
488 67 133 195 246
514 27 53 78 98
561 195 380 555 700
638 no data no data no data no data
Intensity measured (in micro watt) using 60X (oil) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 14 28 63 77
457 0 0 2.3 3
476 3 6 8 10
488 18 35 51 66
514 9 17 26 32
561 73 141 203 261
638 no data no data no data no data
0 : value under detection level
Craig

On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, Andreas. It bothered me for many years that people still claimed that
> a CLSM gives you brighter images when you use a lower magnification
> objective (for the same NA). Physically, it didn't make sense to me. I
> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> If you consider the focused spot on a CLSM, the size of the PSF depends
> only on the NA of the objective and not it's magnification, so the
> illumination will be identical for a 40x and a 63x objective with the same
> NA (assuming that you overfill the back aperture in both cases to take full
> advantage of the NA of the lens). Now consider the detection: again, only
> the NA determines how much light you will collect by the lens. So it
> wouldn’t make any sense for a CLSM to give you a "brighter" image with a
> lower mag lens when both lenses have the same NA.
>
> But wait! When you look into the binocular it looks brighter with the 40x
> lens. AND, if you keep all of the same settings (laser power percentage
> and detector gain) you get a brighter image with the 40x objective. So
> what's going on? My relatively new Thorlabs power meter (PM400 console
> with S170C sensor) is compatible with oil immersion and the difference in
> brightness with the 40x objective is 100% accounted for by the change in
> laser power when you switch between these objectives. The change in laser
> power is due to the smaller back aperture of the 63x objective. In other
> words, when you switch from the 40x to the 63x objective, the edges of the
> laser beam are blocked by the smaller aperture of the 63x lens, so less
> excitation reaches the sample. If you adjust the % laser power slider so
> that both the 40x and 63x objectives are reading the same illumination
> intensity, then you get the exact same image with both lenses.
>
> As you mentioned, I tried to explain this in our Nat Prot paper in
> Supplementary Figure 1 and I included some of the data there (free download
> for the Supp Figs - for the full paper if anyone needs it I'm happy to
> email it to them).
> https://www.nature.com/articles/s41596-020-0313-9
>
> So why is this so broadly misunderstood (I have heard it many, many
> times!)? When we read the classic textbooks on the brightness of a
> microscope image, these were originally written with respect to
> transmitted-light brightfield microscopy: it's not obvious that they should
> apply to confocal microscopy or even to widefield fluorescence microscopy.
> On the Microscopy Primer website (
> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> example, they start with the typical statement that the Image Brightness is
> proportional to (NA/M)^2. They go on to mention that for fluorescence the
> Image Brightness should be lambda NA^4/ M^2. However, they fail to mention
> that the reason for the Mag being in the denominator of the equation is
> because the size of the back aperature depends on Mag in this way. So even
> for a widefield fluorescence microscope, the increase in brightness is
> caused by increased illumination on the sample, not increased detection
> efficiency, which is not very helpful in this era of over-powered
> fluorescence lamps.
>
> If the confocal manufacturers would specify their laser powers in
> real-world units instead of %_of_maximum, when you switch lenses you would
> immediately see that that for a given excitation power density (in W/cm^2)
> you get the same intensity image for 2 lenses with the same NA, regardless
> of the mag of the lens.
>
> Cheers,
> James
>
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> www.aomf.ca<http://www.aomf.ca>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [hidden email]
> Subject: [External] Re: [EXT] Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Hi Andreas,
>
> If you divide the same amount of light across a more magnified PSF, then
> the PSF covers more pixels and so each pixel gets fewer photons. However,
> in this case you would also be more densely sampled, and you could
> digitally downsample the image, which would have the effect of putting the
> same number photons into fewer pixels. If dark and read noise are low,
> this would effectively give you the same image as you would have gotten
> using a lower magnification to begin with.
>
> Mike
>
> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> > 65dOAWbgN2OxNnKaw&e=
> > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> > qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > and include the link in your posting.
> > *****
> >
> > Dear all,
> > Are lower magnification objectives brighter than higher magnification
> > ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> > to 63x NA 1.4? I mean for confocal microscopy.
> >
> > Confocal.nl stated this is a recent webinar and on their website:
> > “A lower magnification allows for a larger field of view and brighter
> > images, since light intensity is inversely proportional to the
> > magnification squared”
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >
> > I would think that this is caused by less light going through the
> > smaller back focal aperture when the illumination is held constant?
> > Most of the light is clipped as explained in fig 1 of
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> > nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > illumination beam path and laser powers to best suit the objective?Or
> > are lower magnification objectives really brighter?
> >
> > The field of view will obviously be larger for the 40x objective, but
> > I am more interested to understand the claimed benefit in brightness.
> >
> > best wishes
> >
> > Andreas
> >
>
> This e-mail may contain confidential and/or privileged information for the
> sole use of the intended recipient.
> Any review or distribution by anyone other than the person for whom it was
> originally intended is strictly prohibited.
> If you have received this e-mail in error, please contact the sender and
> delete all copies.
> Opinions, conclusions or other information contained in this e-mail may
> not be that of the organization.
>
> If you feel you have received an email from UHN of a commercial nature and
> would like to be removed from the sender's mailing list please do one of
> the following:
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> (2) Where no unsubscribe process has been included, reply to the sender
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>
>
> Patient Consent for Email:
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> UHN patients may provide their consent to communicate with UHN about their
> care using email. All electronic communication carries some risk. Please
> visit our website here<
> https://www.uhn.ca/PatientsFamilies/Patient_Safety_Advocacy/Privacy/Documents/Email_consent_and_safety.pdf>
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>

Craig Brideau

Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Those power readings were taken years ago actually and I've since replaced
the argon with a 488 diode laser. We didn't need the extra lines argon
provides on that particular scope. Now on the other hand our upright system
has had its argon retubed twice and is still trucking along because we
needed 488, 457 and 514 nm on that one.

Craig

On Mon, Mar 22, 2021 at 2:03 PM MICROSCOPIA IBIS <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Craig.
> I wonder your drastical decrease power in your Argon laser in 514 line
> respect to 488 one. We had similar problem in one of the Ar laser we had
> with warranty and they had to change the laser. The power of 514 nm line
> has not to be too small than the 488, I think (
> https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1
> )
> [
> https://i1.rgstatic.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/links/09e4150f7f8c30a99c000000/largepreview.png
> ]<
> https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1
> >
> (PDF) Design and implementation of a sensitive high-resolution nonlinear
> spectral imaging microscope<
> https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1
> >
> PDF | Live tissue nonlinear microscopy based on multiphoton
> autofluorescence and second harmonic emission originating from endogenous
> fluorophores and... | Find, read and cite all the research you need on
> ResearchGate
> www.researchgate.net
> Regards,
> Konstantin
>
> ________________________________
> De: Confocal Microscopy List <[hidden email]> en nombre
> de Craig Brideau <[hidden email]>
> Enviado: lunes, 22 de marzo de 2021 20:00
> Para: [hidden email] <[hidden email]>
> Asunto: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for the great answer James! For additional information, here's some
> power readings from one of our confocals at various wavelengths. As you can
> see between the 20x and 60x there is considerable variability by laser
> color as well as by magnification. Units are in microwatts.
> Intensity measured (in micro watt) using 20X (air) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 78 233 400 555
> 457 4 7 10 11
> 476 10 19 27 35
> 488 67 133 195 246
> 514 27 53 78 98
> 561 195 380 555 700
> 638 no data no data no data no data
> Intensity measured (in micro watt) using 60X (oil) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 14 28 63 77
> 457 0 0 2.3 3
> 476 3 6 8 10
> 488 18 35 51 66
> 514 9 17 26 32
> 561 73 141 203 261
> 638 no data no data no data no data
> 0 : value under detection level
> Craig
>
> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi, Andreas. It bothered me for many years that people still claimed
> that
> > a CLSM gives you brighter images when you use a lower magnification
> > objective (for the same NA). Physically, it didn't make sense to me. I
> > have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> > If you consider the focused spot on a CLSM, the size of the PSF depends
> > only on the NA of the objective and not it's magnification, so the
> > illumination will be identical for a 40x and a 63x objective with the
> same
> > NA (assuming that you overfill the back aperture in both cases to take
> full
> > advantage of the NA of the lens). Now consider the detection: again,
> only
> > the NA determines how much light you will collect by the lens. So it
> > wouldn’t make any sense for a CLSM to give you a "brighter" image with a
> > lower mag lens when both lenses have the same NA.
> >
> > But wait! When you look into the binocular it looks brighter with the
> 40x
> > lens. AND, if you keep all of the same settings (laser power percentage
> > and detector gain) you get a brighter image with the 40x objective. So
> > what's going on? My relatively new Thorlabs power meter (PM400 console
> > with S170C sensor) is compatible with oil immersion and the difference in
> > brightness with the 40x objective is 100% accounted for by the change in
> > laser power when you switch between these objectives. The change in
> laser
> > power is due to the smaller back aperture of the 63x objective. In other
> > words, when you switch from the 40x to the 63x objective, the edges of
> the
> > laser beam are blocked by the smaller aperture of the 63x lens, so less
> > excitation reaches the sample. If you adjust the % laser power slider so
> > that both the 40x and 63x objectives are reading the same illumination
> > intensity, then you get the exact same image with both lenses.
> >
> > As you mentioned, I tried to explain this in our Nat Prot paper in
> > Supplementary Figure 1 and I included some of the data there (free
> download
> > for the Supp Figs - for the full paper if anyone needs it I'm happy to
> > email it to them).
> > https://www.nature.com/articles/s41596-020-0313-9
> >
> > So why is this so broadly misunderstood (I have heard it many, many
> > times!)? When we read the classic textbooks on the brightness of a
> > microscope image, these were originally written with respect to
> > transmitted-light brightfield microscopy: it's not obvious that they
> should
> > apply to confocal microscopy or even to widefield fluorescence
> microscopy.
> > On the Microscopy Primer website (
> > https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> > example, they start with the typical statement that the Image Brightness
> is
> > proportional to (NA/M)^2. They go on to mention that for fluorescence
> the
> > Image Brightness should be lambda NA^4/ M^2. However, they fail to
> mention
> > that the reason for the Mag being in the denominator of the equation is
> > because the size of the back aperature depends on Mag in this way. So
> even
> > for a widefield fluorescence microscope, the increase in brightness is
> > caused by increased illumination on the sample, not increased detection
> > efficiency, which is not very helpful in this era of over-powered
> > fluorescence lamps.
> >
> > If the confocal manufacturers would specify their laser powers in
> > real-world units instead of %_of_maximum, when you switch lenses you
> would
> > immediately see that that for a given excitation power density (in
> W/cm^2)
> > you get the same intensity image for 2 lenses with the same NA,
> regardless
> > of the mag of the lens.
> >
> > Cheers,
> > James
> >
> >
> > -----------------------------------------------
> > James Jonkman, Staff Scientist
> > Advanced Optical Microscopy Facility (AOMF)
> > and Wright Cell Imaging Facility (WCIF)
> > University Health Network
> > MaRS, PMCRT tower, 101 College St., Room 15-305
> > Toronto, ON, CANADA M5G 1L7
> > [hidden email] Tel: 416-581-8593
> > www.aomf.ca<http://www.aomf.ca>
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Michael Giacomelli
> > Sent: Monday, March 22, 2021 1:10 PM
> > To: [hidden email]
> > Subject: [External] Re: [EXT] Are lower magnification objectives
> brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
> > [lists[.]umn[.]edu] Post images on
> >
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> > [imgur[.]com] and include the link in your posting.
> > *****
> >
> > Hi Andreas,
> >
> > If you divide the same amount of light across a more magnified PSF, then
> > the PSF covers more pixels and so each pixel gets fewer photons.
> However,
> > in this case you would also be more densely sampled, and you could
> > digitally downsample the image, which would have the effect of putting
> the
> > same number photons into fewer pixels. If dark and read noise are low,
> > this would effectively give you the same image as you would have gotten
> > using a lower magnification to begin with.
> >
> > Mike
> >
> > On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> > > 65dOAWbgN2OxNnKaw&e=
> > > Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> > > qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Dear all,
> > > Are lower magnification objectives brighter than higher magnification
> > > ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> > > to 63x NA 1.4? I mean for confocal microscopy.
> > >
> > > Confocal.nl stated this is a recent webinar and on their website:
> > > “A lower magnification allows for a larger field of view and brighter
> > > images, since light intensity is inversely proportional to the
> > > magnification squared”
> > > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> > > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> > >
> > > I would think that this is caused by less light going through the
> > > smaller back focal aperture when the illumination is held constant?
> > > Most of the light is clipped as explained in fig 1 of
> > > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> > > nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > > illumination beam path and laser powers to best suit the objective?Or
> > > are lower magnification objectives really brighter?
> > >
> > > The field of view will obviously be larger for the 40x objective, but
> > > I am more interested to understand the claimed benefit in brightness.
> > >
> > > best wishes
> > >
> > > Andreas
> > >
> >
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mmodel

Re: EXT: Re: Are lower magnification objectives brighter?

In reply to this post by Konstantín Levitskiy

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists
> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cm
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> eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3
> 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&res
> erved=0 Post images on
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> *****
>
> Hi, Andreas. It bothered me for many years that people still claimed
> that a CLSM gives you brighter images when you use a lower
> magnification objective (for the same NA). Physically, it didn't make
> sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> If you consider the focused spot on a CLSM, the size of the PSF
> depends only on the NA of the objective and not it's magnification, so
> the illumination will be identical for a 40x and a 63x objective with
> the same NA (assuming that you overfill the back aperture in both
> cases to take full advantage of the NA of the lens). Now consider the
> detection: again, only the NA determines how much light you will
> collect by the lens. So it wouldn't make any sense for a CLSM to give
> you a "brighter" image with a lower mag lens when both lenses have the same NA.
>
> But wait! When you look into the binocular it looks brighter with the
> 40x lens. AND, if you keep all of the same settings (laser power
> percentage and detector gain) you get a brighter image with the 40x
> objective. So what's going on? My relatively new Thorlabs power
> meter (PM400 console with S170C sensor) is compatible with oil
> immersion and the difference in brightness with the 40x objective is
> 100% accounted for by the change in laser power when you switch
> between these objectives. The change in laser power is due to the
> smaller back aperture of the 63x objective. In other words, when you
> switch from the 40x to the 63x objective, the edges of the laser beam
> are blocked by the smaller aperture of the 63x lens, so less
> excitation reaches the sample. If you adjust the % laser power slider
> so that both the 40x and 63x objectives are reading the same illumination intensity, then you get the exact same image with both lenses.
>
> As you mentioned, I tried to explain this in our Nat Prot paper in
> Supplementary Figure 1 and I included some of the data there (free
> download for the Supp Figs - for the full paper if anyone needs it I'm
> happy to email it to them).
> https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%40
> KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd
> 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIjoi
> MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reserve
> d=0
>
> So why is this so broadly misunderstood (I have heard it many, many
> times!)? When we read the classic textbooks on the brightness of a
> microscope image, these were originally written with respect to
> transmitted-light brightfield microscopy: it's not obvious that they
> should apply to confocal microscopy or even to widefield fluorescence microscopy.
> On the Microscopy Primer website (
> https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7C0
> 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec
> 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZ
> sb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3
> D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&am
> p;reserved=0 ), for example, they start with the typical statement
> that the Image Brightness is proportional to (NA/M)^2. They go on to
> mention that for fluorescence the Image Brightness should be lambda
> NA^4/ M^2. However, they fail to mention that the reason for the Mag being in the denominator of the equation is because the size of the back aperature depends on Mag in this way. So even for a widefield fluorescence microscope, the increase in brightness is caused by increased illumination on the sample, not increased detection efficiency, which is not very helpful in this era of over-powered fluorescence lamps.
>
> If the confocal manufacturers would specify their laser powers in
> real-world units instead of %_of_maximum, when you switch lenses you
> would immediately see that that for a given excitation power density
> (in W/cm^2) you get the same intensity image for 2 lenses with the
> same NA, regardless of the mag of the lens.
>
> Cheers,
> James
>
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
>
> https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.a
> omf.ca%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a2550
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> pqrLCFoREVxEIHTzs%3D&reserved=0<https://nam11.safelinks.protection
> .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmode
> l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73
> e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJW
> IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserved=
> 0>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [hidden email]
> Subject: [External] Re: [EXT] Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld
> efense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dc
> onfocalmicroscopy__%3B!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8
> corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm%24&data=04%7C01%7Cmmodel%40KENT
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> SVLc6Bztp0%3D&reserved=0 [imgur[.]com] and include the link in
> your posting.
> *****
>
> Hi Andreas,
>
> If you divide the same amount of light across a more magnified PSF,
> then the PSF covers more pixels and so each pixel gets fewer photons.
> However, in this case you would also be more densely sampled, and you
> could digitally downsample the image, which would have the effect of
> putting the same number photons into fewer pixels. If dark and read
> noise are low, this would effectively give you the same image as you
> would have gotten using a lower magnification to begin with.
>
> Mike
>
> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
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> >
> > Dear all,
> > Are lower magnification objectives brighter than higher
> > magnification ones when they have the same NA, e.g. a 40x NA 1.4
> > objective compared to 63x NA 1.4? I mean for confocal microscopy.
> >
> > Confocal.nl stated this is a recent webinar and on their website:
> > "A lower magnification allows for a larger field of view and
> > brighter images, since light intensity is inversely proportional to
> > the magnification squared"
> > https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur
> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl_-
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN%2
> > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF
> > _z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpE
> > kt GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >
> > I would think that this is caused by less light going through the
> > smaller back focal aperture when the illumination is held constant?
> > Most of the light is clipped as explained in fig 1 of
> > https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur
> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_ar
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SAs%
> > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHB
> > eT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnP
> > uV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ
> > 63 nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > illumination beam path and laser powers to best suit the
> > objective?Or are lower magnification objectives really brighter?
> >
> > The field of view will obviously be larger for the 40x objective,
> > but I am more interested to understand the claimed benefit in brightness.
> >
> > best wishes
> >
> > Andreas
> >
>
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CAUTION: EXTERNAL SENDER Do not click any links, open any attachments, or REPLY to the message unless you trust the sender and know the content is safe.

Jonkman, James

Re: [External] Re: EXT: Re: Are lower magnification objectives brighter?

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Great point, Mike. And thanks also to Craig for the additional data comparing intensities between 20x dry and 63x oil. I feel very comfortable comparing 2 lenses with the same NA and both of them oil immersion, but I'm not certain that the Thorlabs power meter (despite being compatible with oil) necessarily captures 100% of the laser power from the oil objective. The highest angle rays may not hit the sensor - it's difficult to know - and that may account for some of the decrease. This is why I try to benchmark the laser powers using a 10x dry objective on all of my microscopes. Some of the decrease may also be because of lower transmission through the oil objective which has more glass presumably (I liked the recent posts about objective lens designs by the way!). But the bulk of the decrease is because of the smaller back aperture of the 63x objective. I'm also very careful to stop the beam from scanning when I make these measurements. The actual laser power is considerably higher than what you measure when the beam is scanning, simply because the confocal AOTFs blank the beam on fly-back and when changing direction. Choosing a higher zoom doesn't help - you really need to do a point scanning or point bleaching to get the actual power measurement.

Cheers,
James

-----------------------------------------------
James Jonkman, Staff Scientist
Advanced Optical Microscopy Facility (AOMF)
and Wright Cell Imaging Facility (WCIF)
University Health Network
MaRS, PMCRT tower, 101 College St., Room 15-305
Toronto, ON, CANADA M5G 1L7
[hidden email] Tel: 416-581-8593
www.aomf.ca

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Model, Michael
Sent: Tuesday, March 23, 2021 9:11 AM
To: [hidden email]
Subject: [External] Re: EXT: Re: Are lower magnification objectives brighter?

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James,

Since you mentioned transmission bright-field, it is also a widely misunderstood topic because brightness is determined mostly by direct light from the condenser and not by diffracted light, so NA of the objective does not matter at al as long as it is larger than NA of the condenser. In other words, brightness is determined by the smallest NA between the objective and condenser. This can be easily verified using an objective with variable NA. Misstatements on this subject can be found even in some of the classical treatises.

Mike

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of MICROSCOPIA IBIS
Sent: Monday, March 22, 2021 4:03 PM
To: [hidden email]
Subject: EXT: Re: Are lower magnification objectives brighter?

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Hi Craig.
I wonder your drastical decrease power in your Argon laser in 514 line respect to 488 one. We had similar problem in one of the Ar laser we had with warranty and they had to change the laser. The power of 514 nm line has not to be too small than the 488, I think (https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=1SrT5lAPVXTyVRO2fOXPeyPtVpCGQ2ftpWWZ*2Bn10NTw*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$ [nam11[.]safelinks[.]protection[.]outlook[.]com])
[https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fi1.rgstatic.net*2Fpublication*2F23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope*2Flinks*2F09e4150f7f8c30a99c000000*2Flargepreview.png&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=GDf3wIGTkz*2Fe6DUToPUwm*2BgAleM9mgGoSVgUZZwBNEI*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J6XOIb_h$ [nam11[.]safelinks[.]protection[.]outlook[.]com]]<https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=1SrT5lAPVXTyVRO2fOXPeyPtVpCGQ2ftpWWZ*2Bn10NTw*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$ [nam11[.]safelinks[.]protection[.]outlook[.]com]>
(PDF) Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope<https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=1SrT5lAPVXTyVRO2fOXPeyPtVpCGQ2ftpWWZ*2Bn10NTw*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$ [nam11[.]safelinks[.]protection[.]outlook[.]com]>
PDF | Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and... | Find, read and cite all the research you need on ResearchGate https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=http*3A*2F*2Fwww.researchgate.net*2F&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=CV7MqVq4iBPh9*2BT*2Fdq7mvsjG*2FSTZjtNojCePdEps8SE*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J77ThSZ2$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
Regards,
Konstantin

________________________________
De: Confocal Microscopy List <[hidden email]> en nombre de Craig Brideau <[hidden email]>
Enviado: lunes, 22 de marzo de 2021 20:00
Para: [hidden email] <[hidden email]>
Asunto: Re: Are lower magnification objectives brighter?

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Thanks for the great answer James! For additional information, here's some power readings from one of our confocals at various wavelengths. As you can see between the 20x and 60x there is considerable variability by laser color as well as by magnification. Units are in microwatts.
Intensity measured (in micro watt) using 20X (air) objective Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 78 233 400 555
457 4 7 10 11
476 10 19 27 35
488 67 133 195 246
514 27 53 78 98
561 195 380 555 700
638 no data no data no data no data
Intensity measured (in micro watt) using 60X (oil) objective Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 14 28 63 77
457 0 0 2.3 3
476 3 6 8 10
488 18 35 51 66
514 9 17 26 32
561 73 141 203 261
638 no data no data no data no data
0 : value under detection level
Craig

On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James < [hidden email]> wrote:

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> *****
>
> Hi, Andreas. It bothered me for many years that people still claimed
> that a CLSM gives you brighter images when you use a lower
> magnification objective (for the same NA). Physically, it didn't make
> sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> If you consider the focused spot on a CLSM, the size of the PSF
> depends only on the NA of the objective and not it's magnification, so
> the illumination will be identical for a 40x and a 63x objective with
> the same NA (assuming that you overfill the back aperture in both
> cases to take full advantage of the NA of the lens). Now consider the
> detection: again, only the NA determines how much light you will
> collect by the lens. So it wouldn't make any sense for a CLSM to give
> you a "brighter" image with a lower mag lens when both lenses have the same NA.
>
> But wait! When you look into the binocular it looks brighter with the
> 40x lens. AND, if you keep all of the same settings (laser power
> percentage and detector gain) you get a brighter image with the 40x
> objective. So what's going on? My relatively new Thorlabs power
> meter (PM400 console with S170C sensor) is compatible with oil
> immersion and the difference in brightness with the 40x objective is
> 100% accounted for by the change in laser power when you switch
> between these objectives. The change in laser power is due to the
> smaller back aperture of the 63x objective. In other words, when you
> switch from the 40x to the 63x objective, the edges of the laser beam
> are blocked by the smaller aperture of the 63x lens, so less
> excitation reaches the sample. If you adjust the % laser power slider
> so that both the 40x and 63x objectives are reading the same illumination intensity, then you get the exact same image with both lenses.
>
> As you mentioned, I tried to explain this in our Nat Prot paper in
> Supplementary Figure 1 and I included some of the data there (free
> download for the Supp Figs - for the full paper if anyone needs it I'm
> happy to email it to them).
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$ [nam11[.]safelinks[.]protection[.]outlook[.]com].
> nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%40
> KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd
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> sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reserve
> d=0
>
> So why is this so broadly misunderstood (I have heard it many, many
> times!)? When we read the classic textbooks on the brightness of a
> microscope image, these were originally written with respect to
> transmitted-light brightfield microscopy: it's not obvious that they
> should apply to confocal microscopy or even to widefield fluorescence microscopy.
> On the Microscopy Primer website (
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$ [nam11[.]safelinks[.]protection[.]outlook[.]com].
> microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7C0
> 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec
> 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZ
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> D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&am
> p;reserved=0 ), for example, they start with the typical statement
> that the Image Brightness is proportional to (NA/M)^2. They go on to
> mention that for fluorescence the Image Brightness should be lambda
> NA^4/ M^2. However, they fail to mention that the reason for the Mag being in the denominator of the equation is because the size of the back aperature depends on Mag in this way. So even for a widefield fluorescence microscope, the increase in brightness is caused by increased illumination on the sample, not increased detection efficiency, which is not very helpful in this era of over-powered fluorescence lamps.
>
> If the confocal manufacturers would specify their laser powers in
> real-world units instead of %_of_maximum, when you switch lenses you
> would immediately see that that for a given excitation power density
> (in W/cm^2) you get the same intensity image for 2 lenses with the
> same NA, regardless of the mag of the lens.
>
> Cheers,
> James
>
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
>
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>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [hidden email]
> Subject: [External] Re: [EXT] Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7
> Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnkno
> wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiL
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> SVLc6Bztp0%3D&reserved=0 [imgur[.]com] and include the link in
> your posting.
> *****
>
> Hi Andreas,
>
> If you divide the same amount of light across a more magnified PSF,
> then the PSF covers more pixels and so each pixel gets fewer photons.
> However, in this case you would also be more densely sampled, and you
> could digitally downsample the image, which would have the effect of
> putting the same number photons into fewer pixels. If dark and read
> noise are low, this would effectively give you the same image as you
> would have gotten using a lower magnification to begin with.
>
> Mike
>
> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
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> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-
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> > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5o
> > fM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=
> > aB
> > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEo
> > S-
> > 65dOAWbgN2OxNnKaw&e=
> > Post images on
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> > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyi
> > sI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7C
> > dv qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > and include the link in your posting.
> > *****
> >
> > Dear all,
> > Are lower magnification objectives brighter than higher
> > magnification ones when they have the same NA, e.g. a 40x NA 1.4
> > objective compared to 63x NA 1.4? I mean for confocal microscopy.
> >
> > Confocal.nl stated this is a recent webinar and on their website:
> > "A lower magnification allows for a larger field of view and
> > brighter images, since light intensity is inversely proportional to
> > the magnification squared"
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl_-
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
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> > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF
> > _z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpE
> > kt GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >
> > I would think that this is caused by less light going through the
> > smaller back focal aperture when the illumination is held constant?
> > Most of the light is clipped as explained in fig 1 of
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_ar
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SAs%
> > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHB
> > eT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnP
> > uV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ
> > 63 nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > illumination beam path and laser powers to best suit the
> > objective?Or are lower magnification objectives really brighter?
> >
> > The field of view will obviously be larger for the 40x objective,
> > but I am more interested to understand the claimed benefit in brightness.
> >
> > best wishes
> >
> > Andreas
> >
>
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Re: [External] Re: EXT: Re: Are lower magnification objectives brighter?

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On Tue, Mar 23, 2021 at 7:25 AM Jonkman, James <[hidden email]>
wrote:

> I'm not certain that the Thorlabs power meter (despite being compatible
> with oil) necessarily captures 100% of the laser power from the oil
> objective. The highest angle rays may not hit the sensor - it's difficult
> to know - and that may account for some of the decrease.
>

Pina Colarruso and I actually helped Thorlabs develop that power meter, and
our big contribution to the design was ensuring it does, in fact, take into
account the high angle rays. There is a thin layer of index matching
material underneath the glass window that eases the high-angle light into
the silicone detector (*n* = 3 !!!) underneath the window. You can test
this yourself by measuring a high NA oil lens with and without oil on the
sensor.

Dr. Colarruso and I also recently got roped into volunteered to take part
in the QUAREP microscopy initiative for repeatably quantifying microscope
outputs among different systems. Working Group 1 revolves around power
measurements and a very patient Thorlabs engineer has helped explain some
of the fundamentals of the sensor to the group.
Links:
https://quarep.org/
https://quarep.org/working-groups/wg-1-illumination-power/

Craig

>
> Cheers,
> James
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Model, Michael
> Sent: Tuesday, March 23, 2021 9:11 AM
> To: [hidden email]
> Subject: [External] Re: EXT: Re: Are lower magnification objectives
> brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
> James,
>
> Since you mentioned transmission bright-field, it is also a widely
> misunderstood topic because brightness is determined mostly by direct light
> from the condenser and not by diffracted light, so NA of the objective does
> not matter at al as long as it is larger than NA of the condenser. In other
> words, brightness is determined by the smallest NA between the objective
> and condenser. This can be easily verified using an objective with variable
> NA. Misstatements on this subject can be found even in some of the
> classical treatises.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of MICROSCOPIA IBIS
> Sent: Monday, March 22, 2021 4:03 PM
> To: [hidden email]
> Subject: EXT: Re: Are lower magnification objectives brighter?
>
> *****
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>
> Hi Craig.
> I wonder your drastical decrease power in your Argon laser in 514 line
> respect to 488 one. We had similar problem in one of the Ar laser we had
> with warranty and they had to change the laser. The power of 514 nm line
> has not to be too small than the 488, I think (
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> autofluorescence and second harmonic emission originating from endogenous
> fluorophores and... | Find, read and cite all the research you need on
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> Regards,
> Konstantin
>
> ________________________________
> De: Confocal Microscopy List <[hidden email]> en nombre
> de Craig Brideau <[hidden email]>
> Enviado: lunes, 22 de marzo de 2021 20:00
> Para: [hidden email] <[hidden email]>
> Asunto: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
>
> Thanks for the great answer James! For additional information, here's some
> power readings from one of our confocals at various wavelengths. As you can
> see between the 20x and 60x there is considerable variability by laser
> color as well as by magnification. Units are in microwatts.
> Intensity measured (in micro watt) using 20X (air) objective Percentage of
> laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 78 233 400 555
> 457 4 7 10 11
> 476 10 19 27 35
> 488 67 133 195 246
> 514 27 53 78 98
> 561 195 380 555 700
> 638 no data no data no data no data
> Intensity measured (in micro watt) using 60X (oil) objective Percentage of
> laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 14 28 63 77
> 457 0 0 2.3 3
> 476 3 6 8 10
> 488 18 35 51 66
> 514 9 17 26 32
> 561 73 141 203 261
> 638 no data no data no data no data
> 0 : value under detection level
> Craig
>
> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
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> > *****
> >
> > Hi, Andreas. It bothered me for many years that people still claimed
> > that a CLSM gives you brighter images when you use a lower
> > magnification objective (for the same NA). Physically, it didn't make
> > sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss
> LSM700 confocal.
> > If you consider the focused spot on a CLSM, the size of the PSF
> > depends only on the NA of the objective and not it's magnification, so
> > the illumination will be identical for a 40x and a 63x objective with
> > the same NA (assuming that you overfill the back aperture in both
> > cases to take full advantage of the NA of the lens). Now consider the
> > detection: again, only the NA determines how much light you will
> > collect by the lens. So it wouldn't make any sense for a CLSM to give
> > you a "brighter" image with a lower mag lens when both lenses have the
> same NA.
> >
> > But wait! When you look into the binocular it looks brighter with the
> > 40x lens. AND, if you keep all of the same settings (laser power
> > percentage and detector gain) you get a brighter image with the 40x
> > objective. So what's going on? My relatively new Thorlabs power
> > meter (PM400 console with S170C sensor) is compatible with oil
> > immersion and the difference in brightness with the 40x objective is
> > 100% accounted for by the change in laser power when you switch
> > between these objectives. The change in laser power is due to the
> > smaller back aperture of the 63x objective. In other words, when you
> > switch from the 40x to the 63x objective, the edges of the laser beam
> > are blocked by the smaller aperture of the 63x lens, so less
> > excitation reaches the sample. If you adjust the % laser power slider
> > so that both the 40x and 63x objectives are reading the same
> illumination intensity, then you get the exact same image with both lenses.
> >
> > As you mentioned, I tried to explain this in our Nat Prot paper in
> > Supplementary Figure 1 and I included some of the data there (free
> > download for the Supp Figs - for the full paper if anyone needs it I'm
> > happy to email it to them).
> >
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%40
> > KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd
> > 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIjoi
> > MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> > sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reserve
> > d=0
> >
> > So why is this so broadly misunderstood (I have heard it many, many
> > times!)? When we read the classic textbooks on the brightness of a
> > microscope image, these were originally written with respect to
> > transmitted-light brightfield microscopy: it's not obvious that they
> > should apply to confocal microscopy or even to widefield fluorescence
> microscopy.
> > On the Microscopy Primer website (
> >
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook.com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7C0
> > 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec
> > 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZ
> > sb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3
> > D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&am
> > p;reserved=0 ), for example, they start with the typical statement
> > that the Image Brightness is proportional to (NA/M)^2. They go on to
> > mention that for fluorescence the Image Brightness should be lambda
> > NA^4/ M^2. However, they fail to mention that the reason for the Mag
> being in the denominator of the equation is because the size of the back
> aperature depends on Mag in this way. So even for a widefield fluorescence
> microscope, the increase in brightness is caused by increased illumination
> on the sample, not increased detection efficiency, which is not very
> helpful in this era of over-powered fluorescence lamps.
> >
> > If the confocal manufacturers would specify their laser powers in
> > real-world units instead of %_of_maximum, when you switch lenses you
> > would immediately see that that for a given excitation power density
> > (in W/cm^2) you get the same intensity image for 2 lenses with the
> > same NA, regardless of the mag of the lens.
> >
> > Cheers,
> > James
> >
> >
> > -----------------------------------------------
> > James Jonkman, Staff Scientist
> > Advanced Optical Microscopy Facility (AOMF)
> > and Wright Cell Imaging Facility (WCIF)
> > University Health Network
> > MaRS, PMCRT tower, 101 College St., Room 15-305
> > Toronto, ON, CANADA M5G 1L7
> > [hidden email] Tel: 416-581-8593
> >
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> > Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1J3rm7zaj$
> > [nam11[.]safelinks[.]protection]
> > .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmode
> > l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73
> > e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJW
> > IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> > amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserved=
> > 0>
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of Michael Giacomelli
> > Sent: Monday, March 22, 2021 1:10 PM
> > To: [hidden email]
> > Subject: [External] Re: [EXT] Are lower magnification objectives
> brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Furld__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnp
> > A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > efense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dc
> > onfocalmicroscopy__%3B!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8
> > corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm%24&data=04%7C01%7Cmmodel%40KENT
> > .EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f2
> > 6134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4w
> > LjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdat
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> > [lists[.]umn[.]edu] Post images on
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> > A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > efense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!cVq_1LwAt
> > t5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60%24&amp
> > ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7
> > Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnkno
> > wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiL
> > CJXVCI6Mn0%3D%7C3000&sdata=Xkk%2BxZYn%2Fp1oYe%2BcPSDxVVjjyaom5GpOF
> > SVLc6Bztp0%3D&reserved=0 [imgur[.]com] and include the link in
> > your posting.
> > *****
> >
> > Hi Andreas,
> >
> > If you divide the same amount of light across a more magnified PSF,
> > then the PSF covers more pixels and so each pixel gets fewer photons.
> > However, in this case you would also be more densely sampled, and you
> > could digitally downsample the image, which would have the effect of
> > putting the same number photons into fewer pixels. If dark and read
> > noise are low, this would effectively give you the same image as you
> > would have gotten using a lower magnification to begin with.
> >
> > Mike
> >
> > On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
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> > > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5o
> > > fM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=
> > > aB
> > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEo
> > > S-
> > > 65dOAWbgN2OxNnKaw&e=
> > > Post images on
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
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> > > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%
> > > 3DDw&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8
> > > ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63752040249034
> > > 8759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJ
> > > BTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gD6IgbED16AsVzP5GvSPq0W
> > > sRALiXOnn1PZWkjxF9yY%3D&reserved=0
> > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyi
> > > sI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7C
> > > dv qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Dear all,
> > > Are lower magnification objectives brighter than higher
> > > magnification ones when they have the same NA, e.g. a 40x NA 1.4
> > > objective compared to 63x NA 1.4? I mean for confocal microscopy.
> > >
> > > Confocal.nl stated this is a recent webinar and on their website:
> > > "A lower magnification allows for a larger field of view and
> > > brighter images, since light intensity is inversely proportional to
> > > the magnification squared"
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl_-
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
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> > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN%2
> > > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF
> > > _z
> > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpE
> > > kt GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> > >
> > > I would think that this is caused by less light going through the
> > > smaller back focal aperture when the illumination is held constant?
> > > Most of the light is clipped as explained in fig 1 of
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_ar
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SAs%
> > > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHB
> > > eT
> > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnP
> > > uV
> > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ
> > > 63 nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > > illumination beam path and laser powers to best suit the
> > > objective?Or are lower magnification objectives really brighter?
> > >
> > > The field of view will obviously be larger for the 40x objective,
> > > but I am more interested to understand the claimed benefit in
> brightness.
> > >
> > > best wishes
> > >
> > > Andreas
> > >
> >
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Jonkman, James

Re: [External] Re: EXT: Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

That's fantastic, Craig! Kudos to you and Pina - this has become an essential tool in my toolkit (no commercial interest!). I'm sure Pina has mentioned it to me before that the two of you had a hand in this design but I had forgotten.

Cheers,
James

-----------------------------------------------
James Jonkman, Staff Scientist
Advanced Optical Microscopy Facility (AOMF)
and Wright Cell Imaging Facility (WCIF)
University Health Network
MaRS, PMCRT tower, 101 College St., Room 15-305
Toronto, ON, CANADA M5G 1L7
[hidden email] Tel: 416-581-8593
www.aomf.ca

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Tuesday, March 23, 2021 1:30 PM
To: [hidden email]
Subject: Re: [External] Re: EXT: Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

On Tue, Mar 23, 2021 at 7:25 AM Jonkman, James <[hidden email]>
wrote:

> I'm not certain that the Thorlabs power meter (despite being
> compatible with oil) necessarily captures 100% of the laser power from
> the oil objective. The highest angle rays may not hit the sensor -
> it's difficult to know - and that may account for some of the decrease.
>

Pina Colarruso and I actually helped Thorlabs develop that power meter, and our big contribution to the design was ensuring it does, in fact, take into account the high angle rays. There is a thin layer of index matching material underneath the glass window that eases the high-angle light into the silicone detector (*n* = 3 !!!) underneath the window. You can test this yourself by measuring a high NA oil lens with and without oil on the sensor.

Dr. Colarruso and I also recently got roped into volunteered to take part in the QUAREP microscopy initiative for repeatably quantifying microscope outputs among different systems. Working Group 1 revolves around power measurements and a very patient Thorlabs engineer has helped explain some of the fundamentals of the sensor to the group.
Links:
https://urldefense.com/v3/__https://quarep.org/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4_oydvdU$ [quarep[.]org] https://urldefense.com/v3/__https://quarep.org/working-groups/wg-1-illumination-power/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4xiLzN6p$ [quarep[.]org]

Craig

>
> Cheers,
> James
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> http://www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Model, Michael
> Sent: Tuesday, March 23, 2021 9:11 AM
> To: [hidden email]
> Subject: [External] Re: EXT: Re: Are lower magnification objectives
> brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confoca
> lmicroscopy__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHn
> gml3WfaZWN7Rk1J8J-epuF$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!eEhOfVie8
> uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J0kz5YNE$
> [imgur[.]com] and include the link in your posting.
> *****
>
> James,
>
> Since you mentioned transmission bright-field, it is also a widely
> misunderstood topic because brightness is determined mostly by direct
> light from the condenser and not by diffracted light, so NA of the
> objective does not matter at al as long as it is larger than NA of the
> condenser. In other words, brightness is determined by the smallest NA
> between the objective and condenser. This can be easily verified using
> an objective with variable NA. Misstatements on this subject can be
> found even in some of the classical treatises.
>
> Mike
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of MICROSCOPIA IBIS
> Sent: Monday, March 22, 2021 4:03 PM
> To: [hidden email]
> Subject: EXT: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> Q$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> Post images on
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> Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwuIlFdM$
> [nam11[.]safelinks[.]protection[.]outlook[.]com] and include the link
> in your posting.
> *****
>
> Hi Craig.
> I wonder your drastical decrease power in your Argon laser in 514 line
> respect to 488 one. We had similar problem in one of the Ar laser we
> had with warranty and they had to change the laser. The power of 514
> nm line has not to be too small than the 488, I think (
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
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> [nam11[.]safelinks[.]protection[.]outlook[.]com])
> [
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> .com/?url=https*3A*2F*2Fi1.rgstatic.net*2Fpublication*2F23485392_Desig
> n_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral
> _imaging_microscope*2Flinks*2F09e4150f7f8c30a99c000000*2Flargepreview.
> png&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6
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> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
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> [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> (PDF) Design and implementation of a sensitive high-resolution
> nonlinear spectral imaging microscope<
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> .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
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> 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> PDF | Live tissue nonlinear microscopy based on multiphoton
> autofluorescence and second harmonic emission originating from
> endogenous fluorophores and... | Find, read and cite all the research
> you need on ResearchGate
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> [nam11[.]safelinks[.]protection[.]outlook[.]com]
> Regards,
> Konstantin
>
> ________________________________
> De: Confocal Microscopy List <[hidden email]> en
> nombre de Craig Brideau <[hidden email]>
> Enviado: lunes, 22 de marzo de 2021 20:00
> Para: [hidden email]
> <[hidden email]>
> Asunto: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> lapt$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> Post images on
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> [nam11[.]safelinks[.]protection[.]outlook[.]com] and include the link
> in your posting.
> *****
>
> Thanks for the great answer James! For additional information, here's
> some power readings from one of our confocals at various wavelengths.
> As you can see between the 20x and 60x there is considerable
> variability by laser color as well as by magnification. Units are in microwatts.
> Intensity measured (in micro watt) using 20X (air) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 78 233 400 555
> 457 4 7 10 11
> 476 10 19 27 35
> 488 67 133 195 246
> 514 27 53 78 98
> 561 195 380 555 700
> 638 no data no data no data no data
> Intensity measured (in micro watt) using 60X (oil) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 14 28 63 77
> 457 0 0 2.3 3
> 476 3 6 8 10
> 488 18 35 51 66
> 514 9 17 26 32
> 561 73 141 203 261
> 638 no data no data no data no data
> 0 : value under detection level
> Craig
>
> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > ok
> > .com/?url=http*3A*2F*2Flists__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwAiKrY_$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7
> > Cm
> > model%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d
> > 01
> > 8f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3
> > d8
> > eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7
> > C3
> > 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&r
> > es
> > erved=0 Post images on
> >
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> in your posting.
> > *****
> >
> > Hi, Andreas. It bothered me for many years that people still
> > claimed that a CLSM gives you brighter images when you use a lower
> > magnification objective (for the same NA). Physically, it didn't
> > make sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the
> > same Zeiss
> LSM700 confocal.
> > If you consider the focused spot on a CLSM, the size of the PSF
> > depends only on the NA of the objective and not it's magnification,
> > so the illumination will be identical for a 40x and a 63x objective
> > with the same NA (assuming that you overfill the back aperture in
> > both cases to take full advantage of the NA of the lens). Now
> > consider the
> > detection: again, only the NA determines how much light you will
> > collect by the lens. So it wouldn't make any sense for a CLSM to
> > give you a "brighter" image with a lower mag lens when both lenses
> > have the
> same NA.
> >
> > But wait! When you look into the binocular it looks brighter with
> > the 40x lens. AND, if you keep all of the same settings (laser
> > power percentage and detector gain) you get a brighter image with
> > the 40x objective. So what's going on? My relatively new Thorlabs
> > power meter (PM400 console with S170C sensor) is compatible with oil
> > immersion and the difference in brightness with the 40x objective is
> > 100% accounted for by the change in laser power when you switch
> > between these objectives. The change in laser power is due to the
> > smaller back aperture of the 63x objective. In other words, when
> > you switch from the 40x to the 63x objective, the edges of the laser
> > beam are blocked by the smaller aperture of the 63x lens, so less
> > excitation reaches the sample. If you adjust the % laser power
> > slider so that both the 40x and 63x objectives are reading the same
> illumination intensity, then you get the exact same image with both lenses.
> >
> > As you mentioned, I tried to explain this in our Nat Prot paper in
> > Supplementary Figure 1 and I included some of the data there (free
> > download for the Supp Figs - for the full paper if anyone needs it
> > I'm happy to email it to them).
> >
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%
> > 40
> > KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7
> > dd
> > 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIj
> > oi
> > MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&am
> > p;
> > sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reser
> > ve
> > d=0
> >
> > So why is this so broadly misunderstood (I have heard it many, many
> > times!)? When we read the classic textbooks on the brightness of a
> > microscope image, these were originally written with respect to
> > transmitted-light brightfield microscopy: it's not obvious that they
> > should apply to confocal microscopy or even to widefield
> > fluorescence
> microscopy.
> > On the Microscopy Primer website (
> >
> https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7
> > C0
> > 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1
> > ec
> > 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpb
> > GZ
> > sb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0
> > %3
> > D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&
> > am
> > p;reserved=0 ), for example, they start with the typical statement
> > that the Image Brightness is proportional to (NA/M)^2. They go on
> > to mention that for fluorescence the Image Brightness should be
> > lambda NA^4/ M^2. However, they fail to mention that the reason for
> > the Mag
> being in the denominator of the equation is because the size of the
> back aperature depends on Mag in this way. So even for a widefield
> fluorescence microscope, the increase in brightness is caused by
> increased illumination on the sample, not increased detection
> efficiency, which is not very helpful in this era of over-powered fluorescence lamps.
> >
> > If the confocal manufacturers would specify their laser powers in
> > real-world units instead of %_of_maximum, when you switch lenses you
> > would immediately see that that for a given excitation power density
> > (in W/cm^2) you get the same intensity image for 2 lenses with the
> > same NA, regardless of the mag of the lens.
> >
> > Cheers,
> > James
> >
> >
> > -----------------------------------------------
> > James Jonkman, Staff Scientist
> > Advanced Optical Microscopy Facility (AOMF)
> > and Wright Cell Imaging Facility (WCIF)
> > University Health Network
> > MaRS, PMCRT tower, 101 College St., Room 15-305
> > Toronto, ON, CANADA M5G 1L7
> > [hidden email] Tel: 416-581-8593
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > ok
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> > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J-PhdIG6$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > omf.ca%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25
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> > Xs
> > pqrLCFoREVxEIHTzs%3D&reserved=0<https://urldefense.com/v3/__https:
> > //nam11.safelinks.protection__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA2
> > 6V Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1J3rm7zaj$
> > [nam11[.]safelinks[.]protection]
> > .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmo
> > de
> > l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f
> > 73
> > e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8ey
> > JW
> > IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C300
> > 0&
> > amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserve
> > d=
> > 0>
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of Michael Giacomelli
> > Sent: Monday, March 22, 2021 1:10 PM
> > To: [hidden email]
> > Subject: [External] Re: [EXT] Are lower magnification objectives
> brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > ok
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> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
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> > Dc
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> > NT
> > .EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15
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> > 6134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC
> > 4w
> > LjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sd
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> > ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0
> > %7
> > Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnk
> > no
> > wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWw
> > iL
> > CJXVCI6Mn0%3D%7C3000&sdata=Xkk%2BxZYn%2Fp1oYe%2BcPSDxVVjjyaom5Gp
> > OF
> > SVLc6Bztp0%3D&reserved=0 [imgur[.]com] and include the link in
> > your posting.
> > *****
> >
> > Hi Andreas,
> >
> > If you divide the same amount of light across a more magnified PSF,
> > then the PSF covers more pixels and so each pixel gets fewer photons.
> > However, in this case you would also be more densely sampled, and
> > you could digitally downsample the image, which would have the
> > effect of putting the same number photons into fewer pixels. If
> > dark and read noise are low, this would effectively give you the
> > same image as you would have gotten using a lower magnification to begin with.
> >
> > Mike
> >
> > On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > lo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cg
> > > i-
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > 6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > 59
> > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > iI
> > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=355%2FOydVsjZ%2Fyb05O7GUO
> > > gH
> > > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh
> > > 5o
> > > fM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&
> > > m=
> > > aB
> > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUD
> > > Eo
> > > S-
> > > 65dOAWbgN2OxNnKaw&e=
> > > Post images on
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > lo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26
> > > d%
> > > 3DDw&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508
> > > d8
> > > ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490
> > > 34
> > > 8759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiL
> > > CJ
> > > BTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gD6IgbED16AsVzP5GvSPq
> > > 0W
> > > sRALiXOnn1PZWkjxF9yY%3D&reserved=0
> > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGG
> > > yi
> > > sI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D
> > > 7C dv qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Dear all,
> > > Are lower magnification objectives brighter than higher
> > > magnification ones when they have the same NA, e.g. a 40x NA 1.4
> > > objective compared to 63x NA 1.4? I mean for confocal microscopy.
> > >
> > > Confocal.nl stated this is a recent webinar and on their website:
> > > "A lower magnification allows for a larger field of view and
> > > brighter images, since light intensity is inversely proportional
> > > to the magnification squared"
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > lo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl
> > > _-
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > 6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > 59
> > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > iI
> > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN
> > > %2
> > > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0L
> > > yF
> > > _z
> > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZC
> > > pE kt
> > > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> > >
> > > I would think that this is caused by less light going through the
> > > smaller back focal aperture when the illumination is held constant?
> > > Most of the light is clipped as explained in fig 1 of
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > lo
> > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_
> > > ar
> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > 6d
> > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > 59
> > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > iI
> > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SA
> > > s%
> > > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HB
> > > eT
> > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > > nP
> > > uV
> > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_C
> > > yQ
> > > 63 nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust
> > > the illumination beam path and laser powers to best suit the
> > > objective?Or are lower magnification objectives really brighter?
> > >
> > > The field of view will obviously be larger for the 40x objective,
> > > but I am more interested to understand the claimed benefit in
> brightness.
> > >
> > > best wishes
> > >
> > > Andreas
> > >
> >
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> 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > uhn.ca%2FPatientsFamilies%2FPatient_Safety_Advocacy%2FPrivacy%2FDocu
> > me
> > nts%2FEmail_consent_and_safety.pdf&data=04%7C01%7Cmmodel%40KENT.
> > ED
> > U%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26
> > 13
> > 4%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wL
> > jA
> > wMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata
> > =O LT4qqVppV23PhWrwzzCwziYyhBG6pcKK5EnOCrurBE%3D&reserved=0>
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> If you feel you have received an email from UHN of a commercial nature
> and would like to be removed from the sender's mailing list please do
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Craig Brideau

Re: [External] Re: EXT: Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

The key is that by adding the index matching gel under the window, the
fundamental limit of the sensor becomes the angle of incidence at the first
surface of the window. Since this window is a normal coverslip with index
1.5, any light entering the sensor is what a typical sample under a
coverslip would actually receive. An oil lens removes this interface by
using a drop of oil to eliminate the air-glass interface. Now the light is
propagating at a high angle inside the window, where it encounters the
index gel on the way out. If the gel wasn't there, there would be a
glass-air interface on the exit that would reflect some of the light back
into the window. Instead, the gel allows the light to exit the window where
it can then hit the silicon surface of the detector. I had to sit down with
Snell's law and the Fresnel equations to work this out for myself but the
solution is quite elegant.

Craig

On Tue, Mar 23, 2021 at 11:41 AM Jonkman, James <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> That's fantastic, Craig! Kudos to you and Pina - this has become an
> essential tool in my toolkit (no commercial interest!). I'm sure Pina has
> mentioned it to me before that the two of you had a hand in this design but
> I had forgotten.
>
> Cheers,
> James
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Craig Brideau
> Sent: Tuesday, March 23, 2021 1:30 PM
> To: [hidden email]
> Subject: Re: [External] Re: EXT: Re: Are lower magnification objectives
> brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr46wn4a6N$
> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr45XO6fPP$
> [imgur[.]com] and include the link in your posting.
> *****
>
> On Tue, Mar 23, 2021 at 7:25 AM Jonkman, James <
> [hidden email]>
> wrote:
>
> > I'm not certain that the Thorlabs power meter (despite being
> > compatible with oil) necessarily captures 100% of the laser power from
> > the oil objective. The highest angle rays may not hit the sensor -
> > it's difficult to know - and that may account for some of the decrease.
> >
>
> Pina Colarruso and I actually helped Thorlabs develop that power meter,
> and our big contribution to the design was ensuring it does, in fact, take
> into account the high angle rays. There is a thin layer of index matching
> material underneath the glass window that eases the high-angle light into
> the silicone detector (*n* = 3 !!!) underneath the window. You can test
> this yourself by measuring a high NA oil lens with and without oil on the
> sensor.
>
> Dr. Colarruso and I also recently got roped into volunteered to take part
> in the QUAREP microscopy initiative for repeatably quantifying microscope
> outputs among different systems. Working Group 1 revolves around power
> measurements and a very patient Thorlabs engineer has helped explain some
> of the fundamentals of the sensor to the group.
> Links:
>
> https://urldefense.com/v3/__https://quarep.org/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4_oydvdU$
> [quarep[.]org]
> https://urldefense.com/v3/__https://quarep.org/working-groups/wg-1-illumination-power/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4xiLzN6p$
> [quarep[.]org]
>
> Craig
>
>
>
> >
> > Cheers,
> > James
> >
> > -----------------------------------------------
> > James Jonkman, Staff Scientist
> > Advanced Optical Microscopy Facility (AOMF)
> > and Wright Cell Imaging Facility (WCIF)
> > University Health Network
> > MaRS, PMCRT tower, 101 College St., Room 15-305
> > Toronto, ON, CANADA M5G 1L7
> > [hidden email] Tel: 416-581-8593
> > http://www.aomf.ca
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:[hidden email]]
> > On Behalf Of Model, Michael
> > Sent: Tuesday, March 23, 2021 9:11 AM
> > To: [hidden email]
> > Subject: [External] Re: EXT: Re: Are lower magnification objectives
> > brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confoca
> > lmicroscopy__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHn
> > gml3WfaZWN7Rk1J8J-epuF$
> > [lists[.]umn[.]edu] Post images on
> > https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!eEhOfVie8
> > uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J0kz5YNE$
> > [imgur[.]com] and include the link in your posting.
> > *****
> >
> > James,
> >
> > Since you mentioned transmission bright-field, it is also a widely
> > misunderstood topic because brightness is determined mostly by direct
> > light from the condenser and not by diffracted light, so NA of the
> > objective does not matter at al as long as it is larger than NA of the
> > condenser. In other words, brightness is determined by the smallest NA
> > between the objective and condenser. This can be easily verified using
> > an objective with variable NA. Misstatements on this subject can be
> > found even in some of the classical treatises.
> >
> > Mike
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> > Behalf Of MICROSCOPIA IBIS
> > Sent: Monday, March 22, 2021 4:03 PM
> > To: [hidden email]
> > Subject: EXT: Re: Are lower magnification objectives brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
> > roscopy&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d
> > 8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490328
> > 768*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTi
> > I6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=VmXFqmYQUMMiSEfoWiEXjkcH5fBR
> > qmXzguxlBrq6ZYA*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7
> > IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J9t4q2A
> > Q$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > Post images on
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=04*7C01*7Cmmodel*40KE
> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490328768*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
> > ata=QDc8B8mhmO*2BnENMhORNeP7DXKGuAf6Kicyahn*2B8*2BWL0*3D&reserved=
> > 0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26V
> > Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwuIlFdM$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com] and include the link
> > in your posting.
> > *****
> >
> > Hi Craig.
> > I wonder your drastical decrease power in your Argon laser in 514 line
> > respect to 488 one. We had similar problem in one of the Ar laser we
> > had with warranty and they had to change the laser. The power of 514
> > nm line has not to be too small than the 488, I think (
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
> > dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIj
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> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com])
> > [
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fi1.rgstatic.net*2Fpublication*2F23485392_Desig
> > n_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral
> > _imaging_microscope*2Flinks*2F09e4150f7f8c30a99c000000*2Flargepreview.
> > png&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6
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> > OIb_h$ [nam11[.]safelinks[.]protection[.]outlook[.]com]]<
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
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> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> > (PDF) Design and implementation of a sensitive high-resolution
> > nonlinear spectral imaging microscope<
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
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> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> > PDF | Live tissue nonlinear microscopy based on multiphoton
> > autofluorescence and second harmonic emission originating from
> > endogenous fluorophores and... | Find, read and cite all the research
> > you need on ResearchGate
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Fwww.researchgate.net*2F&data=04*7C01*7Cmmod
> > el*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f7
> > 3e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJ
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> > served=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UB
> > tnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J77ThSZ2$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > Regards,
> > Konstantin
> >
> > ________________________________
> > De: Confocal Microscopy List <[hidden email]> en
> > nombre de Craig Brideau <[hidden email]>
> > Enviado: lunes, 22 de marzo de 2021 20:00
> > Para: [hidden email]
> > <[hidden email]>
> > Asunto: Re: Are lower magnification objectives brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
> > roscopy&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d
> > 8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338
> > 766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTi
> > I6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=SDWAOFXB3UXBeN1uo*2FfFWOsi3Q
> > T2yJ8vUtUyhxXidc8*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUl!!Cj
> > cC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J7c-
> > lapt$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > Post images on
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=04*7C01*7Cmmodel*40KE
> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
> > ata=6BMEcMW7H26iSwJyEUipERWLEd6wCNglty8oOMQsxc8*3D&reserved=0__;JS
> > UlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds
> > 6dgWEHngml3WfaZWN7Rk1JzJiy9Uz$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com] and include the link
> > in your posting.
> > *****
> >
> > Thanks for the great answer James! For additional information, here's
> > some power readings from one of our confocals at various wavelengths.
> > As you can see between the 20x and 60x there is considerable
> > variability by laser color as well as by magnification. Units are in
> microwatts.
> > Intensity measured (in micro watt) using 20X (air) objective
> > Percentage of laser used:
> > Wavelength of the laser 25% 50% 75% 100%
> > 408 78 233 400 555
> > 457 4 7 10 11
> > 476 10 19 27 35
> > 488 67 133 195 246
> > 514 27 53 78 98
> > 561 195 380 555 700
> > 638 no data no data no data no data
> > Intensity measured (in micro watt) using 60X (oil) objective
> > Percentage of laser used:
> > Wavelength of the laser 25% 50% 75% 100%
> > 408 14 28 63 77
> > 457 0 0 2.3 3
> > 476 3 6 8 10
> > 488 18 35 51 66
> > 514 9 17 26 32
> > 561 73 141 203 261
> > 638 no data no data no data no data
> > 0 : value under detection level
> > Craig
> >
> > On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok
> > > .com/?url=http*3A*2F*2Flists__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwAiKrY_$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7
> > > Cm
> > > model%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d
> > > 01
> > > 8f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3
> > > d8
> > > eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7
> > > C3
> > > 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&r
> > > es
> > > erved=0 Post images on
> > >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=04*7C01*7Cmmodel*40KE
> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
> > ata=6BMEcMW7H26iSwJyEUipERWLEd6wCNglty8oOMQsxc8*3D&reserved=0__;JS
> > UlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds
> > 6dgWEHngml3WfaZWN7Rk1JzJiy9Uz$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com] and include the link
> > in your posting.
> > > *****
> > >
> > > Hi, Andreas. It bothered me for many years that people still
> > > claimed that a CLSM gives you brighter images when you use a lower
> > > magnification objective (for the same NA). Physically, it didn't
> > > make sense to me. I have both a 63x/1.4NA and a 40x/1.4NA on the
> > > same Zeiss
> > LSM700 confocal.
> > > If you consider the focused spot on a CLSM, the size of the PSF
> > > depends only on the NA of the objective and not it's magnification,
> > > so the illumination will be identical for a 40x and a 63x objective
> > > with the same NA (assuming that you overfill the back aperture in
> > > both cases to take full advantage of the NA of the lens). Now
> > > consider the
> > > detection: again, only the NA determines how much light you will
> > > collect by the lens. So it wouldn't make any sense for a CLSM to
> > > give you a "brighter" image with a lower mag lens when both lenses
> > > have the
> > same NA.
> > >
> > > But wait! When you look into the binocular it looks brighter with
> > > the 40x lens. AND, if you keep all of the same settings (laser
> > > power percentage and detector gain) you get a brighter image with
> > > the 40x objective. So what's going on? My relatively new Thorlabs
> > > power meter (PM400 console with S170C sensor) is compatible with oil
> > > immersion and the difference in brightness with the 40x objective is
> > > 100% accounted for by the change in laser power when you switch
> > > between these objectives. The change in laser power is due to the
> > > smaller back aperture of the 63x objective. In other words, when
> > > you switch from the 40x to the 63x objective, the edges of the laser
> > > beam are blocked by the smaller aperture of the 63x lens, so less
> > > excitation reaches the sample. If you adjust the % laser power
> > > slider so that both the 40x and 63x objectives are reading the same
> > illumination intensity, then you get the exact same image with both
> lenses.
> > >
> > > As you mentioned, I tried to explain this in our Nat Prot paper in
> > > Supplementary Figure 1 and I included some of the data there (free
> > > download for the Supp Figs - for the full paper if anyone needs it
> > > I'm happy to email it to them).
> > >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > > nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%
> > > 40
> > > KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7
> > > dd
> > > 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIj
> > > oi
> > > MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&am
> > > p;
> > > sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reser
> > > ve
> > > d=0
> > >
> > > So why is this so broadly misunderstood (I have heard it many, many
> > > times!)? When we read the classic textbooks on the brightness of a
> > > microscope image, these were originally written with respect to
> > > transmitted-light brightfield microscopy: it's not obvious that they
> > > should apply to confocal microscopy or even to widefield
> > > fluorescence
> > microscopy.
> > > On the Microscopy Primer website (
> > >
> > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook
> > .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > > microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7
> > > C0
> > > 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1
> > > ec
> > > 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpb
> > > GZ
> > > sb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0
> > > %3
> > > D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&
> > > am
> > > p;reserved=0 ), for example, they start with the typical statement
> > > that the Image Brightness is proportional to (NA/M)^2. They go on
> > > to mention that for fluorescence the Image Brightness should be
> > > lambda NA^4/ M^2. However, they fail to mention that the reason for
> > > the Mag
> > being in the denominator of the equation is because the size of the
> > back aperature depends on Mag in this way. So even for a widefield
> > fluorescence microscope, the increase in brightness is caused by
> > increased illumination on the sample, not increased detection
> > efficiency, which is not very helpful in this era of over-powered
> fluorescence lamps.
> > >
> > > If the confocal manufacturers would specify their laser powers in
> > > real-world units instead of %_of_maximum, when you switch lenses you
> > > would immediately see that that for a given excitation power density
> > > (in W/cm^2) you get the same intensity image for 2 lenses with the
> > > same NA, regardless of the mag of the lens.
> > >
> > > Cheers,
> > > James
> > >
> > >
> > > -----------------------------------------------
> > > James Jonkman, Staff Scientist
> > > Advanced Optical Microscopy Facility (AOMF)
> > > and Wright Cell Imaging Facility (WCIF)
> > > University Health Network
> > > MaRS, PMCRT tower, 101 College St., Room 15-305
> > > Toronto, ON, CANADA M5G 1L7
> > > [hidden email] Tel: 416-581-8593
> > >
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok
> > > .com/?url=http*3A*2F*2Fwww.a__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J-PhdIG6$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > omf.ca%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25
> > > 50
> > > 8d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63752040249
> > > 03
> > > 48759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLC
> > > JB
> > > TiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=FhVEsqwOD9EyXWBTZbMmVwSa
> > > Xs
> > > pqrLCFoREVxEIHTzs%3D&reserved=0<https://urldefense.com/v3/__https:
> > > //nam11.safelinks.protection__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA2
> > > 6V Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1J3rm7zaj$
> > > [nam11[.]safelinks[.]protection]
> > > .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmo
> > > de
> > > l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f
> > > 73
> > > e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8ey
> > > JW
> > > IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C300
> > > 0&
> > > amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserve
> > > d=
> > > 0>
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List
> > > [mailto:[hidden email]]
> > > On Behalf Of Michael Giacomelli
> > > Sent: Monday, March 22, 2021 1:10 PM
> > > To: [hidden email]
> > > Subject: [External] Re: [EXT] Are lower magnification objectives
> > brighter?
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok
> > > .com/?url=https*3A*2F*2Furld__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > efense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3
> > > Dc
> > > onfocalmicroscopy__%3B!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87
> > > a8
> > > corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm%24&data=04%7C01%7Cmmodel%40KE
> > > NT
> > > .EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15
> > > f2
> > > 6134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC
> > > 4w
> > > LjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sd
> > > at
> > > a=YamE1qZW2OOPk462j1rGa1zJ2O9w8qfDEUdpY2zMbG8%3D&reserved=0
> > > [lists[.]umn[.]edu] Post images on
> > > https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo
> > > ok
> > > .com/?url=https*3A*2F*2Furld__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > efense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!cVq_1Lw
> > > At
> > > t5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60%24&a
> > > mp
> > > ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0
> > > %7
> > > Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnk
> > > no
> > > wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWw
> > > iL
> > > CJXVCI6Mn0%3D%7C3000&sdata=Xkk%2BxZYn%2Fp1oYe%2BcPSDxVVjjyaom5Gp
> > > OF
> > > SVLc6Bztp0%3D&reserved=0 [imgur[.]com] and include the link in
> > > your posting.
> > > *****
> > >
> > > Hi Andreas,
> > >
> > > If you divide the same amount of light across a more magnified PSF,
> > > then the PSF covers more pixels and so each pixel gets fewer photons.
> > > However, in this case you would also be more densely sampled, and
> > > you could digitally downsample the image, which would have the
> > > effect of putting the same number photons into fewer pixels. If
> > > dark and read noise are low, this would effectively give you the
> > > same image as you would have gotten using a lower magnification to
> begin with.
> > >
> > > Mike
> > >
> > > On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> > > [hidden email]> wrote:
> > >
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
> > > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cg
> > > > i-
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=355%2FOydVsjZ%2Fyb05O7GUO
> > > > gH
> > > > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh
> > > > 5o
> > > > fM
> > > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&
> > > > m=
> > > > aB
> > > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUD
> > > > Eo
> > > > S-
> > > > 65dOAWbgN2OxNnKaw&e=
> > > > Post images on
> > > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26
> > > > d%
> > > > 3DDw&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508
> > > > d8
> > > > ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490
> > > > 34
> > > > 8759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiL
> > > > CJ
> > > > BTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gD6IgbED16AsVzP5GvSPq
> > > > 0W
> > > > sRALiXOnn1PZWkjxF9yY%3D&reserved=0
> > > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGG
> > > > yi
> > > > sI
> > > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D
> > > > 7C dv qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > > > and include the link in your posting.
> > > > *****
> > > >
> > > > Dear all,
> > > > Are lower magnification objectives brighter than higher
> > > > magnification ones when they have the same NA, e.g. a 40x NA 1.4
> > > > objective compared to 63x NA 1.4? I mean for confocal microscopy.
> > > >
> > > > Confocal.nl stated this is a recent webinar and on their website:
> > > > "A lower magnification allows for a larger field of view and
> > > > brighter images, since light intensity is inversely proportional
> > > > to the magnification squared"
> > > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl
> > > > _-
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN
> > > > %2
> > > > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0L
> > > > yF
> > > > _z
> > > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZC
> > > > pE kt
> > > > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> > > >
> > > > I would think that this is caused by less light going through the
> > > > smaller back focal aperture when the illumination is held constant?
> > > > Most of the light is clipped as explained in fig 1 of
> > > > https://urldefense.com/v3/__https://nam11.safelinks.protection.out
> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_
> > > > ar
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SA
> > > > s%
> > > > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > > HB
> > > > eT
> > > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > > > nP
> > > > uV
> > > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_C
> > > > yQ
> > > > 63 nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust
> > > > the illumination beam path and laser powers to best suit the
> > > > objective?Or are lower magnification objectives really brighter?
> > > >
> > > > The field of view will obviously be larger for the 40x objective,
> > > > but I am more interested to understand the claimed benefit in
> > brightness.
> > > >
> > > > best wishes
> > > >
> > > > Andreas
> > > >
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> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > > uhn.ca%2FPatientsFamilies%2FPatient_Safety_Advocacy%2FPrivacy%2FDocu
> > > me
> > > nts%2FEmail_consent_and_safety.pdf&data=04%7C01%7Cmmodel%40KENT.
> > > ED
> > > U%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26
> > > 13
> > > 4%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wL
> > > jA
> > > wMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata
> > > =O LT4qqVppV23PhWrwzzCwziYyhBG6pcKK5EnOCrurBE%3D&reserved=0>
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0000001ed7f52e4a-dmarc-request

Re: Are lower magnification objectives brighter?

In reply to this post by Craig Brideau

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Dear James, Craig and others,
Thanks for the detailed explanations, this all makes sense to me now. While this was initially only indented for confocal, I did a simple experiment with the widefield microscope, comparing 20x NA 0.8 and 40x NA 0.75 objectives. The images were taken with the same pixel size (2 times binning on the 40x) and the same region (cropping for the 20x). Same LED power and acquisition time settings. Interestingly, the fluorescence intensity of the larger magnification 40x was 1.8x higher!!! When measuring the LED power, it was 2x higher out of the 20x objective.
I think the 2x higher LED power is spread over a 4x larger area in case of the 20x objective, so that the power density is half compared to the 40x objective, leading to the lower fluorescence intensity of the image with the 20x objective. The difference between the measured 1.8 and 2.0 could be assigned to the difference in NA^2 and probably slight differences in transmission. Does this makes sense?
best wishes
Andreas

-----Original Message-----
From: Craig Brideau <[hidden email]>
To: [hidden email]
Sent: Mon, 22 Mar 2021 19:00
Subject: Re: Are lower magnification objectives brighter?

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Thanks for the great answer James! For additional information, here's some
power readings from one of our confocals at various wavelengths. As you can
see between the 20x and 60x there is considerable variability by laser
color as well as by magnification. Units are in microwatts.
Intensity measured (in micro watt) using 20X (air) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 78 233 400 555
457 4 7 10 11
476 10 19 27 35
488 67 133 195 246
514 27 53 78 98
561 195 380 555 700
638 no data no data no data no data
Intensity measured (in micro watt) using 60X (oil) objective
Percentage of laser used:
Wavelength of the laser 25% 50% 75% 100%
408 14 28 63 77
457 0 0 2.3 3
476 3 6 8 10
488 18 35 51 66
514 9 17 26 32
561 73 141 203 261
638 no data no data no data no data
0 : value under detection level
Craig

On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, Andreas. It bothered me for many years that people still claimed that
> a CLSM gives you brighter images when you use a lower magnification
> objective (for the same NA). Physically, it didn't make sense to me. I
> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> If you consider the focused spot on a CLSM, the size of the PSF depends
> only on the NA of the objective and not it's magnification, so the
> illumination will be identical for a 40x and a 63x objective with the same
> NA (assuming that you overfill the back aperture in both cases to take full
> advantage of the NA of the lens). Now consider the detection: again, only
> the NA determines how much light you will collect by the lens. So it
> wouldn’t make any sense for a CLSM to give you a "brighter" image with a
> lower mag lens when both lenses have the same NA.
>
> But wait! When you look into the binocular it looks brighter with the 40x
> lens. AND, if you keep all of the same settings (laser power percentage
> and detector gain) you get a brighter image with the 40x objective. So
> what's going on? My relatively new Thorlabs power meter (PM400 console
> with S170C sensor) is compatible with oil immersion and the difference in
> brightness with the 40x objective is 100% accounted for by the change in
> laser power when you switch between these objectives. The change in laser
> power is due to the smaller back aperture of the 63x objective. In other
> words, when you switch from the 40x to the 63x objective, the edges of the
> laser beam are blocked by the smaller aperture of the 63x lens, so less
> excitation reaches the sample. If you adjust the % laser power slider so
> that both the 40x and 63x objectives are reading the same illumination
> intensity, then you get the exact same image with both lenses.
>
> As you mentioned, I tried to explain this in our Nat Prot paper in
> Supplementary Figure 1 and I included some of the data there (free download
> for the Supp Figs - for the full paper if anyone needs it I'm happy to
> email it to them).
> https://www.nature.com/articles/s41596-020-0313-9
>
> So why is this so broadly misunderstood (I have heard it many, many
> times!)? When we read the classic textbooks on the brightness of a
> microscope image, these were originally written with respect to
> transmitted-light brightfield microscopy: it's not obvious that they should
> apply to confocal microscopy or even to widefield fluorescence microscopy.
> On the Microscopy Primer website (
> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> example, they start with the typical statement that the Image Brightness is
> proportional to (NA/M)^2. They go on to mention that for fluorescence the
> Image Brightness should be lambda NA^4/ M^2. However, they fail to mention
> that the reason for the Mag being in the denominator of the equation is
> because the size of the back aperature depends on Mag in this way. So even
> for a widefield fluorescence microscope, the increase in brightness is
> caused by increased illumination on the sample, not increased detection
> efficiency, which is not very helpful in this era of over-powered
> fluorescence lamps.
>
> If the confocal manufacturers would specify their laser powers in
> real-world units instead of %_of_maximum, when you switch lenses you would
> immediately see that that for a given excitation power density (in W/cm^2)
> you get the same intensity image for 2 lenses with the same NA, regardless
> of the mag of the lens.
>
> Cheers,
> James
>
>
> -----------------------------------------------
> James Jonkman, Staff Scientist
> Advanced Optical Microscopy Facility (AOMF)
> and Wright Cell Imaging Facility (WCIF)
> University Health Network
> MaRS, PMCRT tower, 101 College St., Room 15-305
> Toronto, ON, CANADA M5G 1L7
> [hidden email] Tel: 416-581-8593
> www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [hidden email]
> Subject: [External] Re: [EXT] Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> [lists[.]umn[.]edu] Post images on
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> [imgur[.]com] and include the link in your posting.
> *****
>
> Hi Andreas,
>
> If you divide the same amount of light across a more magnified PSF, then
> the PSF covers more pixels and so each pixel gets fewer photons. However,
> in this case you would also be more densely sampled, and you could
> digitally downsample the image, which would have the effect of putting the
> same number photons into fewer pixels. If dark and read noise are low,
> this would effectively give you the same image as you would have gotten
> using a lower magnification to begin with.
>
> Mike
>
> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> > 65dOAWbgN2OxNnKaw&e=
> > Post images on
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> > qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > and include the link in your posting.
> > *****
> >
> > Dear all,
> > Are lower magnification objectives brighter than higher magnification
> > ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> > to 63x NA 1.4? I mean for confocal microscopy.
> >
> > Confocal.nl stated this is a recent webinar and on their website:
> > “A lower magnification allows for a larger field of view and brighter
> > images, since light intensity is inversely proportional to the
> > magnification squared”
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >
> > I would think that this is caused by less light going through the
> > smaller back focal aperture when the illumination is held constant?
> > Most of the light is clipped as explained in fig 1 of
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> > nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > illumination beam path and laser powers to best suit the objective?Or
> > are lower magnification objectives really brighter?
> >
> > The field of view will obviously be larger for the 40x objective, but
> > I am more interested to understand the claimed benefit in brightness.
> >
> > best wishes
> >
> > Andreas
> >
>
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Craig Brideau

Re: Are lower magnification objectives brighter?

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Another subtlety when considering camera binning: combining 4 pixels to
make one larger effective pixel loses a bit of light due to the dead space
"+" between the four pixels. CMOS and CCD also handle binning differently
which is something to consider.

Regarding power throughput for objectives: For some higher-end laser
scanning systems there is a variable telescope built into the scanhead on
the excitation input. Our Nikon A1 had this feature for 2P mode. When you
switched lenses it would move the telescope elements and change the beam
diameter to better match the back aperture of the current objective. For
lower mag lenses it would widen the beam, and for higher mag lenses it
would shrink the beam. Simpler systems use a fixed wide beam diameter to
match the worst-case low mag scenario and just eat the power loss when
using a higher mag smaller back aperture lens assuming the NA increase will
help make up the difference.

Craig

On Wed, Mar 24, 2021 at 1:30 PM Andreas Bruckbauer <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear James, Craig and others,
> Thanks for the detailed explanations, this all makes sense to me now.
> While this was initially only indented for confocal, I did a simple
> experiment with the widefield microscope, comparing 20x NA 0.8 and 40x NA
> 0.75 objectives. The images were taken with the same pixel size (2 times
> binning on the 40x) and the same region (cropping for the 20x). Same LED
> power and acquisition time settings. Interestingly, the fluorescence
> intensity of the larger magnification 40x was 1.8x higher!!! When
> measuring the LED power, it was 2x higher out of the 20x objective.
> I think the 2x higher LED power is spread over a 4x larger area in case of
> the 20x objective, so that the power density is half compared to the 40x
> objective, leading to the lower fluorescence intensity of the image with
> the 20x objective. The difference between the measured 1.8 and 2.0 could be
> assigned to the difference in NA^2 and probably slight differences in
> transmission. Does this makes sense?
> best wishes
> Andreas
>
>
> -----Original Message-----
> From: Craig Brideau <[hidden email]>
> To: [hidden email]
> Sent: Mon, 22 Mar 2021 19:00
> Subject: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for the great answer James! For additional information, here's some
> power readings from one of our confocals at various wavelengths. As you can
> see between the 20x and 60x there is considerable variability by laser
> color as well as by magnification. Units are in microwatts.
> Intensity measured (in micro watt) using 20X (air) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 78 233 400 555
> 457 4 7 10 11
> 476 10 19 27 35
> 488 67 133 195 246
> 514 27 53 78 98
> 561 195 380 555 700
> 638 no data no data no data no data
> Intensity measured (in micro watt) using 60X (oil) objective
> Percentage of laser used:
> Wavelength of the laser 25% 50% 75% 100%
> 408 14 28 63 77
> 457 0 0 2.3 3
> 476 3 6 8 10
> 488 18 35 51 66
> 514 9 17 26 32
> 561 73 141 203 261
> 638 no data no data no data no data
> 0 : value under detection level
> Craig
>
> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi, Andreas. It bothered me for many years that people still claimed
> that
> > a CLSM gives you brighter images when you use a lower magnification
> > objective (for the same NA). Physically, it didn't make sense to me. I
> > have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
> > If you consider the focused spot on a CLSM, the size of the PSF depends
> > only on the NA of the objective and not it's magnification, so the
> > illumination will be identical for a 40x and a 63x objective with the
> same
> > NA (assuming that you overfill the back aperture in both cases to take
> full
> > advantage of the NA of the lens). Now consider the detection: again,
> only
> > the NA determines how much light you will collect by the lens. So it
> > wouldn’t make any sense for a CLSM to give you a "brighter" image with a
> > lower mag lens when both lenses have the same NA.
> >
> > But wait! When you look into the binocular it looks brighter with the
> 40x
> > lens. AND, if you keep all of the same settings (laser power percentage
> > and detector gain) you get a brighter image with the 40x objective. So
> > what's going on? My relatively new Thorlabs power meter (PM400 console
> > with S170C sensor) is compatible with oil immersion and the difference in
> > brightness with the 40x objective is 100% accounted for by the change in
> > laser power when you switch between these objectives. The change in
> laser
> > power is due to the smaller back aperture of the 63x objective. In other
> > words, when you switch from the 40x to the 63x objective, the edges of
> the
> > laser beam are blocked by the smaller aperture of the 63x lens, so less
> > excitation reaches the sample. If you adjust the % laser power slider so
> > that both the 40x and 63x objectives are reading the same illumination
> > intensity, then you get the exact same image with both lenses.
> >
> > As you mentioned, I tried to explain this in our Nat Prot paper in
> > Supplementary Figure 1 and I included some of the data there (free
> download
> > for the Supp Figs - for the full paper if anyone needs it I'm happy to
> > email it to them).
> > https://www.nature.com/articles/s41596-020-0313-9
> >
> > So why is this so broadly misunderstood (I have heard it many, many
> > times!)? When we read the classic textbooks on the brightness of a
> > microscope image, these were originally written with respect to
> > transmitted-light brightfield microscopy: it's not obvious that they
> should
> > apply to confocal microscopy or even to widefield fluorescence
> microscopy.
> > On the Microscopy Primer website (
> > https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> > example, they start with the typical statement that the Image Brightness
> is
> > proportional to (NA/M)^2. They go on to mention that for fluorescence
> the
> > Image Brightness should be lambda NA^4/ M^2. However, they fail to
> mention
> > that the reason for the Mag being in the denominator of the equation is
> > because the size of the back aperature depends on Mag in this way. So
> even
> > for a widefield fluorescence microscope, the increase in brightness is
> > caused by increased illumination on the sample, not increased detection
> > efficiency, which is not very helpful in this era of over-powered
> > fluorescence lamps.
> >
> > If the confocal manufacturers would specify their laser powers in
> > real-world units instead of %_of_maximum, when you switch lenses you
> would
> > immediately see that that for a given excitation power density (in
> W/cm^2)
> > you get the same intensity image for 2 lenses with the same NA,
> regardless
> > of the mag of the lens.
> >
> > Cheers,
> > James
> >
> >
> > -----------------------------------------------
> > James Jonkman, Staff Scientist
> > Advanced Optical Microscopy Facility (AOMF)
> > and Wright Cell Imaging Facility (WCIF)
> > University Health Network
> > MaRS, PMCRT tower, 101 College St., Room 15-305
> > Toronto, ON, CANADA M5G 1L7
> > [hidden email] Tel: 416-581-8593
> > www.aomf.ca
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Michael Giacomelli
> > Sent: Monday, March 22, 2021 1:10 PM
> > To: [hidden email]
> > Subject: [External] Re: [EXT] Are lower magnification objectives
> brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
> > [lists[.]umn[.]edu] Post images on
> >
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> > [imgur[.]com] and include the link in your posting.
> > *****
> >
> > Hi Andreas,
> >
> > If you divide the same amount of light across a more magnified PSF, then
> > the PSF covers more pixels and so each pixel gets fewer photons.
> However,
> > in this case you would also be more densely sampled, and you could
> > digitally downsample the image, which would have the effect of putting
> the
> > same number photons into fewer pixels. If dark and read noise are low,
> > this would effectively give you the same image as you would have gotten
> > using a lower magnification to begin with.
> >
> > Mike
> >
> > On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> > [hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> > > 65dOAWbgN2OxNnKaw&e=
> > > Post images on
> > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> > > qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Dear all,
> > > Are lower magnification objectives brighter than higher magnification
> > > ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> > > to 63x NA 1.4? I mean for confocal microscopy.
> > >
> > > Confocal.nl stated this is a recent webinar and on their website:
> > > “A lower magnification allows for a larger field of view and brighter
> > > images, since light intensity is inversely proportional to the
> > > magnification squared”
> > > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> > > GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> > >
> > > I would think that this is caused by less light going through the
> > > smaller back focal aperture when the illumination is held constant?
> > > Most of the light is clipped as explained in fig 1 of
> > > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> > > nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> > > illumination beam path and laser powers to best suit the objective?Or
> > > are lower magnification objectives really brighter?
> > >
> > > The field of view will obviously be larger for the 40x objective, but
> > > I am more interested to understand the claimed benefit in brightness.
> > >
> > > best wishes
> > >
> > > Andreas
> > >
> >
> > This e-mail may contain confidential and/or privileged information for
> the
> > sole use of the intended recipient.
> > Any review or distribution by anyone other than the person for whom it
> was
> > originally intended is strictly prohibited.
> > If you have received this e-mail in error, please contact the sender and
> > delete all copies.
> > Opinions, conclusions or other information contained in this e-mail may
> > not be that of the organization.
> >
> > If you feel you have received an email from UHN of a commercial nature
> and
> > would like to be removed from the sender's mailing list please do one of
> > the following:
> > (1) Follow any unsubscribe process the sender has included in their email
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> > information please go to our UHN Newsletters and Mailing Lists page.
> > Please note that we are unable to automatically unsubscribe individuals
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> >
> >
> > Patient Consent for Email:
> >
> > UHN patients may provide their consent to communicate with UHN about
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> > care using email. All electronic communication carries some risk. Please
> > visit our website here<
> >
> https://www.uhn.ca/PatientsFamilies/Patient_Safety_Advocacy/Privacy/Documents/Email_consent_and_safety.pdf
> >
> > to learn about the risks of electronic communication and how to protect
> > your privacy. You may withdraw your consent to receive emails from UHN at
> > any time. Please contact your care provider or the UHN Privacy Office at
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>

Arnaud ROYON

Re: Are lower magnification objectives brighter?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear list,

/"You can test this yourself by measuring a high NA oil lens with and
without oil on the sensor."/

This test might give you some indications, but I do not think it would
prove that all (or most of) the rays are collected by a power sensor
containing an index-matching gel.
In confocal microscopy, when using an oil objective without oil on the
sensor, there is another interface to consider, where total internal
reflection (TIR) occurs: it is the interface between the objective front
lens and the immersion medium (/i.e./ air if no oil is used).
At this interface, the ray with angles higher than the TIR limit angle
are reflected. This artificially decreases the NA of the objective.

Maybe a more suitable test would be to measure the power coming out of
the oil objective (with oil on the sensor) with a power meter containing
an index-matching gel, and the same power meter with the index-matching
gel removed.

However, going back to the initial topic of this discussion, but from
the NA perspective, it is actually a good way to check how the
brightness evolves with NA.
Take a stable fluorescent sample, ideally just below a cover-slip (to be
in the design configuration of the cover-slip corrected objectives).
Select an oil objective, and image the sample with and without oil.
Magnification remains the same, while numerical aperture changes.
You will directly notice a drop of contrast (due to degraded resolution)
and brightness.
In wide-field microscopy, this is due to TIR at the coverslip / air
interface (rays are coming from the sample to the objective).
In confocal microscopy, this is due to TIR at the objective front lens /
air interface (rays are coming from the objective to the sample).
If you can estimate (from Snell-Descartes calculations for instance) the
objective NA without oil, you can verify the formula "Brightness=NA^4 /
Mag^2 ".
Or, if you can measure the drop of brightness, you can determine the
drop of NA when no oil is put on the coverslip.

From the magnification perspective, as James wrote, take two objectives
with different magnifications and same NA, make sure you have the same
power at the sample plane with a power meter (you do not necessarily
need to measure the absolute values, since you are comparing two
powers), image a stable sample, and check how the brightness evolves
with magnification.

Best regards,
Arnaud

Arnaud ROYON, Ph.D.
CSO & CTO, Member of the Executive Board, Co-founder
Argolight
Cité de la Photonique, Bat. Elnath
11 avenue de Canteranne
33600 Pessac, FRANCE
Email: [hidden email]
Tel: (+33) 5 64 31 08 50
Web site: www.argolight.com

Le 24/03/2021 à 21:12, Craig Brideau a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Another subtlety when considering camera binning: combining 4 pixels to
> make one larger effective pixel loses a bit of light due to the dead space
> "+" between the four pixels. CMOS and CCD also handle binning differently
> which is something to consider.
>
> Regarding power throughput for objectives: For some higher-end laser
> scanning systems there is a variable telescope built into the scanhead on
> the excitation input. Our Nikon A1 had this feature for 2P mode. When you
> switched lenses it would move the telescope elements and change the beam
> diameter to better match the back aperture of the current objective. For
> lower mag lenses it would widen the beam, and for higher mag lenses it
> would shrink the beam. Simpler systems use a fixed wide beam diameter to
> match the worst-case low mag scenario and just eat the power loss when
> using a higher mag smaller back aperture lens assuming the NA increase will
> help make up the difference.
>
> Craig
>
> On Wed, Mar 24, 2021 at 1:30 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear James, Craig and others,
>> Thanks for the detailed explanations, this all makes sense to me now.
>> While this was initially only indented for confocal, I did a simple
>> experiment with the widefield microscope, comparing 20x NA 0.8 and 40x NA
>> 0.75 objectives. The images were taken with the same pixel size (2 times
>> binning on the 40x) and the same region (cropping for the 20x). Same LED
>> power and acquisition time settings. Interestingly, the fluorescence
>> intensity of the larger magnification 40x was 1.8x higher!!! When
>> measuring the LED power, it was 2x higher out of the 20x objective.
>> I think the 2x higher LED power is spread over a 4x larger area in case of
>> the 20x objective, so that the power density is half compared to the 40x
>> objective, leading to the lower fluorescence intensity of the image with
>> the 20x objective. The difference between the measured 1.8 and 2.0 could be
>> assigned to the difference in NA^2 and probably slight differences in
>> transmission. Does this makes sense?
>> best wishes
>> Andreas
>>
>>
>> -----Original Message-----
>> From: Craig Brideau <[hidden email]>
>> To: [hidden email]
>> Sent: Mon, 22 Mar 2021 19:00
>> Subject: Re: Are lower magnification objectives brighter?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Thanks for the great answer James! For additional information, here's some
>> power readings from one of our confocals at various wavelengths. As you can
>> see between the 20x and 60x there is considerable variability by laser
>> color as well as by magnification. Units are in microwatts.
>> Intensity measured (in micro watt) using 20X (air) objective
>> Percentage of laser used:
>> Wavelength of the laser 25% 50% 75% 100%
>> 408 78 233 400 555
>> 457 4 7 10 11
>> 476 10 19 27 35
>> 488 67 133 195 246
>> 514 27 53 78 98
>> 561 195 380 555 700
>> 638 no data no data no data no data
>> Intensity measured (in micro watt) using 60X (oil) objective
>> Percentage of laser used:
>> Wavelength of the laser 25% 50% 75% 100%
>> 408 14 28 63 77
>> 457 0 0 2.3 3
>> 476 3 6 8 10
>> 488 18 35 51 66
>> 514 9 17 26 32
>> 561 73 141 203 261
>> 638 no data no data no data no data
>> 0 : value under detection level
>> Craig
>>
>> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hi, Andreas. It bothered me for many years that people still claimed
>> that
>>> a CLSM gives you brighter images when you use a lower magnification
>>> objective (for the same NA). Physically, it didn't make sense to me. I
>>> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
>>> If you consider the focused spot on a CLSM, the size of the PSF depends
>>> only on the NA of the objective and not it's magnification, so the
>>> illumination will be identical for a 40x and a 63x objective with the
>> same
>>> NA (assuming that you overfill the back aperture in both cases to take
>> full
>>> advantage of the NA of the lens). Now consider the detection: again,
>> only
>>> the NA determines how much light you will collect by the lens. So it
>>> wouldn’t make any sense for a CLSM to give you a "brighter" image with a
>>> lower mag lens when both lenses have the same NA.
>>>
>>> But wait! When you look into the binocular it looks brighter with the
>> 40x
>>> lens. AND, if you keep all of the same settings (laser power percentage
>>> and detector gain) you get a brighter image with the 40x objective. So
>>> what's going on? My relatively new Thorlabs power meter (PM400 console
>>> with S170C sensor) is compatible with oil immersion and the difference in
>>> brightness with the 40x objective is 100% accounted for by the change in
>>> laser power when you switch between these objectives. The change in
>> laser
>>> power is due to the smaller back aperture of the 63x objective. In other
>>> words, when you switch from the 40x to the 63x objective, the edges of
>> the
>>> laser beam are blocked by the smaller aperture of the 63x lens, so less
>>> excitation reaches the sample. If you adjust the % laser power slider so
>>> that both the 40x and 63x objectives are reading the same illumination
>>> intensity, then you get the exact same image with both lenses.
>>>
>>> As you mentioned, I tried to explain this in our Nat Prot paper in
>>> Supplementary Figure 1 and I included some of the data there (free
>> download
>>> for the Supp Figs - for the full paper if anyone needs it I'm happy to
>>> email it to them).
>>> https://www.nature.com/articles/s41596-020-0313-9
>>>
>>> So why is this so broadly misunderstood (I have heard it many, many
>>> times!)? When we read the classic textbooks on the brightness of a
>>> microscope image, these were originally written with respect to
>>> transmitted-light brightfield microscopy: it's not obvious that they
>> should
>>> apply to confocal microscopy or even to widefield fluorescence
>> microscopy.
>>> On the Microscopy Primer website (
>>> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
>>> example, they start with the typical statement that the Image Brightness
>> is
>>> proportional to (NA/M)^2. They go on to mention that for fluorescence
>> the
>>> Image Brightness should be lambda NA^4/ M^2. However, they fail to
>> mention
>>> that the reason for the Mag being in the denominator of the equation is
>>> because the size of the back aperature depends on Mag in this way. So
>> even
>>> for a widefield fluorescence microscope, the increase in brightness is
>>> caused by increased illumination on the sample, not increased detection
>>> efficiency, which is not very helpful in this era of over-powered
>>> fluorescence lamps.
>>>
>>> If the confocal manufacturers would specify their laser powers in
>>> real-world units instead of %_of_maximum, when you switch lenses you
>> would
>>> immediately see that that for a given excitation power density (in
>> W/cm^2)
>>> you get the same intensity image for 2 lenses with the same NA,
>> regardless
>>> of the mag of the lens.
>>>
>>> Cheers,
>>> James
>>>
>>>
>>> -----------------------------------------------
>>> James Jonkman, Staff Scientist
>>> Advanced Optical Microscopy Facility (AOMF)
>>> and Wright Cell Imaging Facility (WCIF)
>>> University Health Network
>>> MaRS, PMCRT tower, 101 College St., Room 15-305
>>> Toronto, ON, CANADA M5G 1L7
>>> [hidden email] Tel: 416-581-8593
>>> www.aomf.ca
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of Michael Giacomelli
>>> Sent: Monday, March 22, 2021 1:10 PM
>>> To: [hidden email]
>>> Subject: [External] Re: [EXT] Are lower magnification objectives
>> brighter?
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
>>>
>> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
>>> [lists[.]umn[.]edu] Post images on
>>>
>> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
>>> [imgur[.]com] and include the link in your posting.
>>> *****
>>>
>>> Hi Andreas,
>>>
>>> If you divide the same amount of light across a more magnified PSF, then
>>> the PSF covers more pixels and so each pixel gets fewer photons.
>> However,
>>> in this case you would also be more densely sampled, and you could
>>> digitally downsample the image, which would have the effect of putting
>> the
>>> same number photons into fewer pixels. If dark and read noise are low,
>>> this would effectively give you the same image as you would have gotten
>>> using a lower magnification to begin with.
>>>
>>> Mike
>>>
>>> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
>>> [hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
>>>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
>>>> HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
>>>> nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
>>>> 65dOAWbgN2OxNnKaw&e=
>>>> Post images on
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
>>>> IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
>>>> eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
>>>> qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
>>>> and include the link in your posting.
>>>> *****
>>>>
>>>> Dear all,
>>>> Are lower magnification objectives brighter than higher magnification
>>>> ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
>>>> to 63x NA 1.4? I mean for confocal microscopy.
>>>>
>>>> Confocal.nl stated this is a recent webinar and on their website:
>>>> “A lower magnification allows for a larger field of view and brighter
>>>> images, since light intensity is inversely proportional to the
>>>> magnification squared”
>>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
>>>> 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
>>>> 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
>>>> GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
>>>>
>>>> I would think that this is caused by less light going through the
>>>> smaller back focal aperture when the illumination is held constant?
>>>> Most of the light is clipped as explained in fig 1 of
>>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
>>>> ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
>>>> l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
>>>> l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
>>>> nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
>>>> illumination beam path and laser powers to best suit the objective?Or
>>>> are lower magnification objectives really brighter?
>>>>
>>>> The field of view will obviously be larger for the 40x objective, but
>>>> I am more interested to understand the claimed benefit in brightness.
>>>>
>>>> best wishes
>>>>
>>>> Andreas
>>>>
>>> This e-mail may contain confidential and/or privileged information for
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Craig Brideau

Re: Are lower magnification objectives brighter?

> /"You can test this yourself by measuring a high NA oil lens with and
> without oil on the sensor."/
> This test might give you some indications, but I do not think it would
> prove that all (or most of) the rays are collected by a power sensor
> containing an index-matching gel.
> In confocal microscopy, when using an oil objective without oil on the
> sensor, there is another interface to consider, where total internal
> reflection (TIR) occurs: it is the interface between the objective front
> lens and the immersion medium (/i.e./ air if no oil is used).
> At this interface, the ray with angles higher than the TIR limit angle
> are reflected. This artificially decreases the NA of the objective.
>

If you work out the Fresnel equations for a few different cases, assuming a
coverglass of index 1.5, you start to see a significant increase in
reflectivity around 61 degrees incidence for air lenses, and about 72
degrees for water lenses. Keep in mind the air lens has an overall higher
reflectivity as more light is lost at the air glass interface even at
normal incidence. I just eyeballed these from a chart I made so you may
want to check yourself. Oil lenses should have a very small difference as
the oil is supposed to index match very closely to the glass. One advantage
of having a standard coverslip built in to the sensor is that the light it
detects is essentially what any sample under a similar coverslip would
receive; the power level is accurate for the dose experienced by the
specimen.
If you wanted to measure the absolute power exiting the lens you would need
to use an integrating sphere type detector, which is a bit impractical to
fit on a microscope stage, although I'm sure it could be done. I question
how useful this information is though as it wouldn't take into account the
impact of the coverslip and the media on the final dose of light
experienced by the sample.

Craig

>
> Maybe a more suitable test would be to measure the power coming out of
> the oil objective (with oil on the sensor) with a power meter containing
> an index-matching gel, and the same power meter with the index-matching
> gel removed.
>
> However, going back to the initial topic of this discussion, but from
> the NA perspective, it is actually a good way to check how the
> brightness evolves with NA.
> Take a stable fluorescent sample, ideally just below a cover-slip (to be
> in the design configuration of the cover-slip corrected objectives).
> Select an oil objective, and image the sample with and without oil.
> Magnification remains the same, while numerical aperture changes.
> You will directly notice a drop of contrast (due to degraded resolution)
> and brightness.
> In wide-field microscopy, this is due to TIR at the coverslip / air
> interface (rays are coming from the sample to the objective).
> In confocal microscopy, this is due to TIR at the objective front lens /
> air interface (rays are coming from the objective to the sample).
> If you can estimate (from Snell-Descartes calculations for instance) the
> objective NA without oil, you can verify the formula "Brightness=NA^4 /
> Mag^2 ".
> Or, if you can measure the drop of brightness, you can determine the
> drop of NA when no oil is put on the coverslip.
>
> From the magnification perspective, as James wrote, take two objectives
> with different magnifications and same NA, make sure you have the same
> power at the sample plane with a power meter (you do not necessarily
> need to measure the absolute values, since you are comparing two
> powers), image a stable sample, and check how the brightness evolves
> with magnification.
>
> Best regards,
> Arnaud
>
> Arnaud ROYON, Ph.D.
> CSO & CTO, Member of the Executive Board, Co-founder
> Argolight
> Cité de la Photonique, Bat. Elnath
> 11 avenue de Canteranne
> 33600 Pessac, FRANCE
> Email: [hidden email]
> Tel: (+33) 5 64 31 08 50
> Web site: www.argolight.com
>
> Le 24/03/2021 à 21:12, Craig Brideau a écrit :
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Another subtlety when considering camera binning: combining 4 pixels to
> > make one larger effective pixel loses a bit of light due to the dead
> space
> > "+" between the four pixels. CMOS and CCD also handle binning differently
> > which is something to consider.
> >
> > Regarding power throughput for objectives: For some higher-end laser
> > scanning systems there is a variable telescope built into the scanhead on
> > the excitation input. Our Nikon A1 had this feature for 2P mode. When you
> > switched lenses it would move the telescope elements and change the beam
> > diameter to better match the back aperture of the current objective. For
> > lower mag lenses it would widen the beam, and for higher mag lenses it
> > would shrink the beam. Simpler systems use a fixed wide beam diameter to
> > match the worst-case low mag scenario and just eat the power loss when
> > using a higher mag smaller back aperture lens assuming the NA increase
> will
> > help make up the difference.
> >
> > Craig
> >
> > On Wed, Mar 24, 2021 at 1:30 PM Andreas Bruckbauer <
> > [hidden email]> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> Dear James, Craig and others,
> >> Thanks for the detailed explanations, this all makes sense to me now.
> >> While this was initially only indented for confocal, I did a simple
> >> experiment with the widefield microscope, comparing 20x NA 0.8 and 40x
> NA
> >> 0.75 objectives. The images were taken with the same pixel size (2 times
> >> binning on the 40x) and the same region (cropping for the 20x). Same LED
> >> power and acquisition time settings. Interestingly, the fluorescence
> >> intensity of the larger magnification 40x was 1.8x higher!!! When
> >> measuring the LED power, it was 2x higher out of the 20x objective.
> >> I think the 2x higher LED power is spread over a 4x larger area in case
> of
> >> the 20x objective, so that the power density is half compared to the 40x
> >> objective, leading to the lower fluorescence intensity of the image with
> >> the 20x objective. The difference between the measured 1.8 and 2.0
> could be
> >> assigned to the difference in NA^2 and probably slight differences in
> >> transmission. Does this makes sense?
> >> best wishes
> >> Andreas
> >>
> >>
> >> -----Original Message-----
> >> From: Craig Brideau <[hidden email]>
> >> To: [hidden email]
> >> Sent: Mon, 22 Mar 2021 19:00
> >> Subject: Re: Are lower magnification objectives brighter?
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> Thanks for the great answer James! For additional information, here's
> some
> >> power readings from one of our confocals at various wavelengths. As you
> can
> >> see between the 20x and 60x there is considerable variability by laser
> >> color as well as by magnification. Units are in microwatts.
> >> Intensity measured (in micro watt) using 20X (air) objective
> >> Percentage of laser used:
> >> Wavelength of the laser 25% 50% 75% 100%
> >> 408 78 233 400 555
> >> 457 4 7 10 11
> >> 476 10 19 27 35
> >> 488 67 133 195 246
> >> 514 27 53 78 98
> >> 561 195 380 555 700
> >> 638 no data no data no data no data
> >> Intensity measured (in micro watt) using 60X (oil) objective
> >> Percentage of laser used:
> >> Wavelength of the laser 25% 50% 75% 100%
> >> 408 14 28 63 77
> >> 457 0 0 2.3 3
> >> 476 3 6 8 10
> >> 488 18 35 51 66
> >> 514 9 17 26 32
> >> 561 73 141 203 261
> >> 638 no data no data no data no data
> >> 0 : value under detection level
> >> Craig
> >>
> >> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
> >> [hidden email]> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> Post images on http://www.imgur.com and include the link in your
> >> posting.
> >>> *****
> >>>
> >>> Hi, Andreas. It bothered me for many years that people still claimed
> >> that
> >>> a CLSM gives you brighter images when you use a lower magnification
> >>> objective (for the same NA). Physically, it didn't make sense to me.
> I
> >>> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700
> confocal.
> >>> If you consider the focused spot on a CLSM, the size of the PSF
> depends
> >>> only on the NA of the objective and not it's magnification, so the
> >>> illumination will be identical for a 40x and a 63x objective with the
> >> same
> >>> NA (assuming that you overfill the back aperture in both cases to take
> >> full
> >>> advantage of the NA of the lens). Now consider the detection: again,
> >> only
> >>> the NA determines how much light you will collect by the lens. So it
> >>> wouldn’t make any sense for a CLSM to give you a "brighter" image with
> a
> >>> lower mag lens when both lenses have the same NA.
> >>>
> >>> But wait! When you look into the binocular it looks brighter with the
> >> 40x
> >>> lens. AND, if you keep all of the same settings (laser power
> percentage
> >>> and detector gain) you get a brighter image with the 40x objective. So
> >>> what's going on? My relatively new Thorlabs power meter (PM400 console
> >>> with S170C sensor) is compatible with oil immersion and the difference
> in
> >>> brightness with the 40x objective is 100% accounted for by the change
> in
> >>> laser power when you switch between these objectives. The change in
> >> laser
> >>> power is due to the smaller back aperture of the 63x objective. In
> other
> >>> words, when you switch from the 40x to the 63x objective, the edges of
> >> the
> >>> laser beam are blocked by the smaller aperture of the 63x lens, so less
> >>> excitation reaches the sample. If you adjust the % laser power slider
> so
> >>> that both the 40x and 63x objectives are reading the same illumination
> >>> intensity, then you get the exact same image with both lenses.
> >>>
> >>> As you mentioned, I tried to explain this in our Nat Prot paper in
> >>> Supplementary Figure 1 and I included some of the data there (free
> >> download
> >>> for the Supp Figs - for the full paper if anyone needs it I'm happy to
> >>> email it to them).
> >>> https://www.nature.com/articles/s41596-020-0313-9
> >>>
> >>> So why is this so broadly misunderstood (I have heard it many, many
> >>> times!)? When we read the classic textbooks on the brightness of a
> >>> microscope image, these were originally written with respect to
> >>> transmitted-light brightfield microscopy: it's not obvious that they
> >> should
> >>> apply to confocal microscopy or even to widefield fluorescence
> >> microscopy.
> >>> On the Microscopy Primer website (
> >>> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
> >>> example, they start with the typical statement that the Image
> Brightness
> >> is
> >>> proportional to (NA/M)^2. They go on to mention that for fluorescence
> >> the
> >>> Image Brightness should be lambda NA^4/ M^2. However, they fail to
> >> mention
> >>> that the reason for the Mag being in the denominator of the equation is
> >>> because the size of the back aperature depends on Mag in this way. So
> >> even
> >>> for a widefield fluorescence microscope, the increase in brightness is
> >>> caused by increased illumination on the sample, not increased detection
> >>> efficiency, which is not very helpful in this era of over-powered
> >>> fluorescence lamps.
> >>>
> >>> If the confocal manufacturers would specify their laser powers in
> >>> real-world units instead of %_of_maximum, when you switch lenses you
> >> would
> >>> immediately see that that for a given excitation power density (in
> >> W/cm^2)
> >>> you get the same intensity image for 2 lenses with the same NA,
> >> regardless
> >>> of the mag of the lens.
> >>>
> >>> Cheers,
> >>> James
> >>>
> >>>
> >>> -----------------------------------------------
> >>> James Jonkman, Staff Scientist
> >>> Advanced Optical Microscopy Facility (AOMF)
> >>> and Wright Cell Imaging Facility (WCIF)
> >>> University Health Network
> >>> MaRS, PMCRT tower, 101 College St., Room 15-305
> >>> Toronto, ON, CANADA M5G 1L7
> >>> [hidden email] Tel: 416-581-8593
> >>> www.aomf.ca
> >>>
> >>>
> >>> -----Original Message-----
> >>> From: Confocal Microscopy List [mailto:
> [hidden email]]
> >>> On Behalf Of Michael Giacomelli
> >>> Sent: Monday, March 22, 2021 1:10 PM
> >>> To: [hidden email]
> >>> Subject: [External] Re: [EXT] Are lower magnification objectives
> >> brighter?
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>>
> >>>
> >>
> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
> >>> [lists[.]umn[.]edu] Post images on
> >>>
> >>
> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
> >>> [imgur[.]com] and include the link in your posting.
> >>> *****
> >>>
> >>> Hi Andreas,
> >>>
> >>> If you divide the same amount of light across a more magnified PSF,
> then
> >>> the PSF covers more pixels and so each pixel gets fewer photons.
> >> However,
> >>> in this case you would also be more densely sampled, and you could
> >>> digitally downsample the image, which would have the effect of putting
> >> the
> >>> same number photons into fewer pixels. If dark and read noise are low,
> >>> this would effectively give you the same image as you would have gotten
> >>> using a lower magnification to begin with.
> >>>
> >>> Mike
> >>>
> >>> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
> >>> [hidden email]> wrote:
> >>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>>
> >>>>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> >>>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> >>>> HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> >>>> nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
> >>>> 65dOAWbgN2OxNnKaw&e=
> >>>> Post images on
> >>>>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
> >>>> IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
> >>>> eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
> >>>> qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
> >>>> and include the link in your posting.
> >>>> *****
> >>>>
> >>>> Dear all,
> >>>> Are lower magnification objectives brighter than higher magnification
> >>>> ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
> >>>> to 63x NA 1.4? I mean for confocal microscopy.
> >>>>
> >>>> Confocal.nl stated this is a recent webinar and on their website:
> >>>> “A lower magnification allows for a larger field of view and brighter
> >>>> images, since light intensity is inversely proportional to the
> >>>> magnification squared”
> >>>>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
> >>>> 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
> >>>> 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
> >>>> GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
> >>>>
> >>>> I would think that this is caused by less light going through the
> >>>> smaller back focal aperture when the illumination is held constant?
> >>>> Most of the light is clipped as explained in fig 1 of
> >>>>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
> >>>> ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
> >>>> l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
> >>>> l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
> >>>> nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
> >>>> illumination beam path and laser powers to best suit the objective?Or
> >>>> are lower magnification objectives really brighter?
> >>>>
> >>>> The field of view will obviously be larger for the 40x objective, but
> >>>> I am more interested to understand the claimed benefit in brightness.
> >>>>
> >>>> best wishes
> >>>>
> >>>> Andreas
> >>>>
> >>> This e-mail may contain confidential and/or privileged information for
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0000001ed7f52e4a-dmarc-request

Re: Are lower magnification objectives brighter?

In reply to this post by Arnaud ROYON

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Hi Arnaud,

make sure you have the same
power at the sample plane with a power meter

You also need to take into account the area which is illuminated. When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
best wishes
Andreas

-----Original Message-----
From: Arnaud ROYON <[hidden email]>
To: [hidden email]
Sent: Thu, 25 Mar 2021 9:38
Subject: Re: Are lower magnification objectives brighter?

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Dear list,

/"You can test this yourself by measuring a high NA oil lens with and
without oil on the sensor."/

This test might give you some indications, but I do not think it would
prove that all (or most of) the rays are collected by a power sensor
containing an index-matching gel.
In confocal microscopy, when using an oil objective without oil on the
sensor, there is another interface to consider, where total internal
reflection (TIR) occurs: it is the interface between the objective front
lens and the immersion medium (/i.e./ air if no oil is used).
At this interface, the ray with angles higher than the TIR limit angle
are reflected. This artificially decreases the NA of the objective.

Maybe a more suitable test would be to measure the power coming out of
the oil objective (with oil on the sensor) with a power meter containing
an index-matching gel, and the same power meter with the index-matching
gel removed.

However, going back to the initial topic of this discussion, but from
the NA perspective, it is actually a good way to check how the
brightness evolves with NA.
Take a stable fluorescent sample, ideally just below a cover-slip (to be
in the design configuration of the cover-slip corrected objectives).
Select an oil objective, and image the sample with and without oil.
Magnification remains the same, while numerical aperture changes.
You will directly notice a drop of contrast (due to degraded resolution)
and brightness.
In wide-field microscopy, this is due to TIR at the coverslip / air
interface (rays are coming from the sample to the objective).
In confocal microscopy, this is due to TIR at the objective front lens /
air interface (rays are coming from the objective to the sample).
If you can estimate (from Snell-Descartes calculations for instance) the
objective NA without oil, you can verify the formula "Brightness=NA^4 /
Mag^2 ".
Or, if you can measure the drop of brightness, you can determine the
drop of NA when no oil is put on the coverslip.

From the magnification perspective, as James wrote, take two objectives
with different magnifications and same NA, make sure you have the same
power at the sample plane with a power meter (you do not necessarily
need to measure the absolute values, since you are comparing two
powers), image a stable sample, and check how the brightness evolves
with magnification.

Best regards,
Arnaud

Arnaud ROYON, Ph.D.
CSO & CTO, Member of the Executive Board, Co-founder
Argolight
Cité de la Photonique, Bat. Elnath
11 avenue de Canteranne
33600 Pessac, FRANCE
Email: [hidden email]
Tel: (+33) 5 64 31 08 50
Web site: www.argolight.com

Le 24/03/2021 à 21:12, Craig Brideau a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Another subtlety when considering camera binning: combining 4 pixels to
> make one larger effective pixel loses a bit of light due to the dead space
> "+" between the four pixels. CMOS and CCD also handle binning differently
> which is something to consider.
>
> Regarding power throughput for objectives: For some higher-end laser
> scanning systems there is a variable telescope built into the scanhead on
> the excitation input. Our Nikon A1 had this feature for 2P mode. When you
> switched lenses it would move the telescope elements and change the beam
> diameter to better match the back aperture of the current objective. For
> lower mag lenses it would widen the beam, and for higher mag lenses it
> would shrink the beam. Simpler systems use a fixed wide beam diameter to
> match the worst-case low mag scenario and just eat the power loss when
> using a higher mag smaller back aperture lens assuming the NA increase will
> help make up the difference.
>
> Craig
>
> On Wed, Mar 24, 2021 at 1:30 PM Andreas Bruckbauer <
> [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear James, Craig and others,
>> Thanks for the detailed explanations, this all makes sense to me now.
>> While this was initially only indented for confocal, I did a simple
>> experiment with the widefield microscope, comparing 20x NA 0.8 and 40x NA
>> 0.75 objectives. The images were taken with the same pixel size (2 times
>> binning on the 40x) and the same region (cropping for the 20x). Same LED
>> power and acquisition time settings. Interestingly, the fluorescence
>> intensity of the larger magnification 40x was 1.8x higher!!! When
>> measuring the LED power, it was 2x higher out of the 20x objective.
>> I think the 2x higher LED power is spread over a 4x larger area in case of
>> the 20x objective, so that the power density is half compared to the 40x
>> objective, leading to the lower fluorescence intensity of the image with
>> the 20x objective. The difference between the measured 1.8 and 2.0 could be
>> assigned to the difference in NA^2 and probably slight differences in
>> transmission. Does this makes sense?
>> best wishes
>> Andreas
>>
>>
>> -----Original Message-----
>> From: Craig Brideau <[hidden email]>
>> To: [hidden email]
>> Sent: Mon, 22 Mar 2021 19:00
>> Subject: Re: Are lower magnification objectives brighter?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Thanks for the great answer James! For additional information, here's some
>> power readings from one of our confocals at various wavelengths. As you can
>> see between the 20x and 60x there is considerable variability by laser
>> color as well as by magnification. Units are in microwatts.
>> Intensity measured (in micro watt) using 20X (air) objective
>> Percentage of laser used:
>> Wavelength of the laser 25% 50% 75% 100%
>> 408 78 233 400 555
>> 457 4 7 10 11
>> 476 10 19 27 35
>> 488 67 133 195 246
>> 514 27 53 78 98
>> 561 195 380 555 700
>> 638 no data no data no data no data
>> Intensity measured (in micro watt) using 60X (oil) objective
>> Percentage of laser used:
>> Wavelength of the laser 25% 50% 75% 100%
>> 408 14 28 63 77
>> 457 0 0 2.3 3
>> 476 3 6 8 10
>> 488 18 35 51 66
>> 514 9 17 26 32
>> 561 73 141 203 261
>> 638 no data no data no data no data
>> 0 : value under detection level
>> Craig
>>
>> On Mon, Mar 22, 2021 at 12:46 PM Jonkman, James <
>> [hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hi, Andreas. It bothered me for many years that people still claimed
>> that
>>> a CLSM gives you brighter images when you use a lower magnification
>>> objective (for the same NA). Physically, it didn't make sense to me. I
>>> have both a 63x/1.4NA and a 40x/1.4NA on the same Zeiss LSM700 confocal.
>>> If you consider the focused spot on a CLSM, the size of the PSF depends
>>> only on the NA of the objective and not it's magnification, so the
>>> illumination will be identical for a 40x and a 63x objective with the
>> same
>>> NA (assuming that you overfill the back aperture in both cases to take
>> full
>>> advantage of the NA of the lens). Now consider the detection: again,
>> only
>>> the NA determines how much light you will collect by the lens. So it
>>> wouldn’t make any sense for a CLSM to give you a "brighter" image with a
>>> lower mag lens when both lenses have the same NA.
>>>
>>> But wait! When you look into the binocular it looks brighter with the
>> 40x
>>> lens. AND, if you keep all of the same settings (laser power percentage
>>> and detector gain) you get a brighter image with the 40x objective. So
>>> what's going on? My relatively new Thorlabs power meter (PM400 console
>>> with S170C sensor) is compatible with oil immersion and the difference in
>>> brightness with the 40x objective is 100% accounted for by the change in
>>> laser power when you switch between these objectives. The change in
>> laser
>>> power is due to the smaller back aperture of the 63x objective. In other
>>> words, when you switch from the 40x to the 63x objective, the edges of
>> the
>>> laser beam are blocked by the smaller aperture of the 63x lens, so less
>>> excitation reaches the sample. If you adjust the % laser power slider so
>>> that both the 40x and 63x objectives are reading the same illumination
>>> intensity, then you get the exact same image with both lenses.
>>>
>>> As you mentioned, I tried to explain this in our Nat Prot paper in
>>> Supplementary Figure 1 and I included some of the data there (free
>> download
>>> for the Supp Figs - for the full paper if anyone needs it I'm happy to
>>> email it to them).
>>> https://www.nature.com/articles/s41596-020-0313-9
>>>
>>> So why is this so broadly misunderstood (I have heard it many, many
>>> times!)? When we read the classic textbooks on the brightness of a
>>> microscope image, these were originally written with respect to
>>> transmitted-light brightfield microscopy: it's not obvious that they
>> should
>>> apply to confocal microscopy or even to widefield fluorescence
>> microscopy.
>>> On the Microscopy Primer website (
>>> https://www.microscopyu.com/microscopy-basics/image-brightness ), for
>>> example, they start with the typical statement that the Image Brightness
>> is
>>> proportional to (NA/M)^2. They go on to mention that for fluorescence
>> the
>>> Image Brightness should be lambda NA^4/ M^2. However, they fail to
>> mention
>>> that the reason for the Mag being in the denominator of the equation is
>>> because the size of the back aperature depends on Mag in this way. So
>> even
>>> for a widefield fluorescence microscope, the increase in brightness is
>>> caused by increased illumination on the sample, not increased detection
>>> efficiency, which is not very helpful in this era of over-powered
>>> fluorescence lamps.
>>>
>>> If the confocal manufacturers would specify their laser powers in
>>> real-world units instead of %_of_maximum, when you switch lenses you
>> would
>>> immediately see that that for a given excitation power density (in
>> W/cm^2)
>>> you get the same intensity image for 2 lenses with the same NA,
>> regardless
>>> of the mag of the lens.
>>>
>>> Cheers,
>>> James
>>>
>>>
>>> -----------------------------------------------
>>> James Jonkman, Staff Scientist
>>> Advanced Optical Microscopy Facility (AOMF)
>>> and Wright Cell Imaging Facility (WCIF)
>>> University Health Network
>>> MaRS, PMCRT tower, 101 College St., Room 15-305
>>> Toronto, ON, CANADA M5G 1L7
>>> [hidden email] Tel: 416-581-8593
>>> www.aomf.ca
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of Michael Giacomelli
>>> Sent: Monday, March 22, 2021 1:10 PM
>>> To: [hidden email]
>>> Subject: [External] Re: [EXT] Are lower magnification objectives
>> brighter?
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
>>>
>> https://urldefense.com/v3/__http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm$
>>> [lists[.]umn[.]edu] Post images on
>>>
>> https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60$
>>> [imgur[.]com] and include the link in your posting.
>>> *****
>>>
>>> Hi Andreas,
>>>
>>> If you divide the same amount of light across a more magnified PSF, then
>>> the PSF covers more pixels and so each pixel gets fewer photons.
>> However,
>>> in this case you would also be more densely sampled, and you could
>>> digitally downsample the image, which would have the effect of putting
>> the
>>> same number photons into fewer pixels. If dark and read noise are low,
>>> this would effectively give you the same image as you would have gotten
>>> using a lower magnification to begin with.
>>>
>>> Mike
>>>
>>> On Mon, Mar 22, 2021 at 1:02 PM Andreas Bruckbauer <
>>> [hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
>>>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
>>>> HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
>>>> nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEoS-
>>>> 65dOAWbgN2OxNnKaw&e=
>>>> Post images on
>>>> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=Dw
>>>> IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
>>>> eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdv
>>>> qg&s=roevs0gDRqIs8bZKBI0bE8ejnEfLkz7n1a9vJZoNMeE&e=
>>>> and include the link in your posting.
>>>> *****
>>>>
>>>> Dear all,
>>>> Are lower magnification objectives brighter than higher magnification
>>>> ones when they have the same NA, e.g. a 40x NA 1.4 objective compared
>>>> to 63x NA 1.4? I mean for confocal microscopy.
>>>>
>>>> Confocal.nl stated this is a recent webinar and on their website:
>>>> “A lower magnification allows for a larger field of view and brighter
>>>> images, since light intensity is inversely proportional to the
>>>> magnification squared”
>>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.confocal.nl_-
>>>> 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z
>>>> 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEkt
>>>> GwklB9D7Cdvqg&s=FRdNlG-gKHQ7Lkl2vBS1jL6SlXxTyAMcF_pCXgVvfao&e=
>>>>
>>>> I would think that this is caused by less light going through the
>>>> smaller back focal aperture when the illumination is held constant?
>>>> Most of the light is clipped as explained in fig 1 of
>>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar
>>>> ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeT
>>>> l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuV
>>>> l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ63
>>>> nsf-C6L2XiGYA&e= So, the microscope manufacturer could adjust the
>>>> illumination beam path and laser powers to best suit the objective?Or
>>>> are lower magnification objectives really brighter?
>>>>
>>>> The field of view will obviously be larger for the 40x objective, but
>>>> I am more interested to understand the claimed benefit in brightness.
>>>>
>>>> best wishes
>>>>
>>>> Andreas
>>>>
>>> This e-mail may contain confidential and/or privileged information for
>> the
>>> sole use of the intended recipient.
>>> Any review or distribution by anyone other than the person for whom it
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Stanislav Vitha-2

Re: Are lower magnification objectives brighter?

In reply to this post by 0000001ed7f52e4a-dmarc-request

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Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.

Stan
Texas A&M University
Microscopy and Imaging Center

On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:

>Hi Arnaud,
>
>make sure you have the same
>power at the sample plane with a power meter
>
>You also need to take into account the area which is illuminated. When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>best wishes
>Andreas
>