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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 <[log in to unmask]>
To: [log in to unmask]
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 <
[log in to unmask]> 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://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
>  [log in to unmask]  Tel: 416-581-8593
>    www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Michael Giacomelli
> Sent: Monday, March 22, 2021 1:10 PM
> To: [log in to unmask]
> 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 <
> [log in to unmask]> 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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