http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592019.html
power readings from one of our confocals at various wavelengths. As you can
color as well as by magnification. Units are in microwatts.
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http://www.imgur.com and include the link in your posting.
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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:
>
> > *****
> > 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
> >
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> > 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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