http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592033.html
"+" between the four pixels. CMOS and CCD also handle binning differently
the excitation input. Our Nikon A1 had this feature for 2P mode. When you
diameter to better match the back aperture of the current objective. For
would shrink the beam. Simpler systems use a fixed wide beam diameter to
> *****
> 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
> > (2) Where no unsubscribe process has been included, reply to the sender
> > and type "unsubscribe" in the subject line. If you require additional
> > information please go to our UHN Newsletters and Mailing Lists page.
> > Please note that we are unable to automatically unsubscribe individuals
> > from all UHN mailing lists.
> >
> >
> > Patient Consent for Email:
> >
> > 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> >
> > 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
> > (416) 340-4800 ext. 6937 if you do not wish to receive emails from UHN.
> >
>