http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592037.html
degrees for water lenses. Keep in mind the air lens has an overall higher
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
fit on a microscope stage, although I'm sure it could be done. I question
>
> 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
> >>>>
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