http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592024.html
> objective. The highest angle rays may not hit the sensor - it's difficult
account the high angle rays. There is a thin layer of index matching
the silicone detector (*n* = 3 !!!) underneath the window. You can test
Dr. Colarruso and I also recently got roped into volunteered to take part
outputs among different systems. Working Group 1 revolves around power
>
> 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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>
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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?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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>
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> Post images on
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> 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 (
>
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> [
>
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>
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> (PDF) Design and implementation of a sensitive high-resolution nonlinear
> spectral imaging microscope<
>
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> 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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> 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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>
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> 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:
> >
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> > 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&res
> > erved=0 Post images on
> >
>
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> 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).
> >
>
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> > 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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> > 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:
> >
> >
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> > 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
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> >
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> > efense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!cVq_1LwAt
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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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> > > S-
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> > > Post images on
> > >
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> > > 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
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> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
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> > > _z
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> > > 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
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> > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
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> > > 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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