http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592021.html
the argon with a 488 diode laser. We didn't need the extra lines argon
provides on that particular scope. Now on the other hand our upright system
> *****
> 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 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 (
>
https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1> )
> [
>
https://i1.rgstatic.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/links/09e4150f7f8c30a99c000000/largepreview.png> ]<
>
https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1> >
> (PDF) Design and implementation of a sensitive high-resolution nonlinear
> spectral imaging microscope<
>
https://www.researchgate.net/publication/23485392_Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral_imaging_microscope/figures?lo=1> >
> 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
> www.researchgate.net
> 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:
>
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<
http://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.
> >
>