http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592026.html
surface of the window. Since this window is a normal coverslip with index
coverslip would actually receive. An oil lens removes this interface by
using a drop of oil to eliminate the air-glass interface. Now the light is
index gel on the way out. If the gel wasn't there, there would be a
into the window. Instead, the gel allows the light to exit the window where
it can then hit the silicon surface of the detector. I had to sit down with
> *****
> 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.
> *****
>
> That's fantastic, Craig! Kudos to you and Pina - this has become an
> essential tool in my toolkit (no commercial interest!). I'm sure Pina has
> mentioned it to me before that the two of you had a hand in this design but
> I had forgotten.
>
> 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 Craig Brideau
> Sent: Tuesday, March 23, 2021 1:30 PM
> To:
[hidden email]
> Subject: Re: [External] Re: EXT: Re: 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!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr46wn4a6N$> [lists[.]umn[.]edu] Post images on
>
https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr45XO6fPP$> [imgur[.]com] and include the link in your posting.
> *****
>
> On Tue, Mar 23, 2021 at 7:25 AM Jonkman, James <
>
[hidden email]>
> wrote:
>
> > I'm not certain that the Thorlabs power meter (despite being
> > compatible with oil) necessarily captures 100% of the laser power from
> > the oil objective. The highest angle rays may not hit the sensor -
> > it's difficult to know - and that may account for some of the decrease.
> >
>
> Pina Colarruso and I actually helped Thorlabs develop that power meter,
> and our big contribution to the design was ensuring it does, in fact, take
> into account the high angle rays. There is a thin layer of index matching
> material underneath the glass window that eases the high-angle light into
> the silicone detector (*n* = 3 !!!) underneath the window. You can test
> this yourself by measuring a high NA oil lens with and without oil on the
> sensor.
>
> Dr. Colarruso and I also recently got roped into volunteered to take part
> in the QUAREP microscopy initiative for repeatably quantifying microscope
> outputs among different systems. Working Group 1 revolves around power
> measurements and a very patient Thorlabs engineer has helped explain some
> of the fundamentals of the sensor to the group.
> Links:
>
>
https://urldefense.com/v3/__https://quarep.org/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4_oydvdU$> [quarep[.]org]
>
https://urldefense.com/v3/__https://quarep.org/working-groups/wg-1-illumination-power/__;!!CjcC7IQ!cbLTSnkhykIT0aO8ynCI7ZT8HFkqSc0R0IgGvmFNetkdvbd8UrtByAGZuxEwoEDr4xiLzN6p$> [quarep[.]org]
>
> Craig
>
>
>
> >
> > 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
> >
http://www.aomf.ca> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> > [mailto:
[hidden email]]
> > On Behalf Of Model, Michael
> > Sent: Tuesday, March 23, 2021 9:11 AM
> > To:
[hidden email]
> > Subject: [External] Re: EXT: Re: 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=confoca> > lmicroscopy__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHn
> > gml3WfaZWN7Rk1J8J-epuF$
> > [lists[.]umn[.]edu] Post images on
> >
https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!eEhOfVie8> > uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J0kz5YNE$
> > [imgur[.]com] and include the link in your posting.
> > *****
> >
> > James,
> >
> > Since you mentioned transmission bright-field, it is also a widely
> > misunderstood topic because brightness is determined mostly by direct
> > light from the condenser and not by diffracted light, so NA of the
> > objective does not matter at al as long as it is larger than NA of the
> > condenser. In other words, brightness is determined by the smallest NA
> > between the objective and condenser. This can be easily verified using
> > an objective with variable NA. Misstatements on this subject can be
> > found even in some of the classical treatises.
> >
> > Mike
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <
[hidden email]> On
> > Behalf Of MICROSCOPIA IBIS
> > Sent: Monday, March 22, 2021 4:03 PM
> > To:
[hidden email]
> > Subject: EXT: Re: Are lower magnification objectives brighter?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
> > roscopy&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d
> > 8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490328
> > 768*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTi
> > I6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=VmXFqmYQUMMiSEfoWiEXjkcH5fBR
> > qmXzguxlBrq6ZYA*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7
> > IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J9t4q2A
> > Q$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > Post images on
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=04*7C01*7Cmmodel*40KE
> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490328768*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
> > ata=QDc8B8mhmO*2BnENMhORNeP7DXKGuAf6Kicyahn*2B8*2BWL0*3D&reserved=
> > 0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26V
> > Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwuIlFdM$
> > [nam11[.]safelinks[.]protection[.]outlook[.]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://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
> > dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIj
> > oiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&am
> > p;sdata=1SrT5lAPVXTyVRO2fOXPeyPtVpCGQ2ftpWWZ*2Bn10NTw*3D&reserved=
> > 0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com])
> > [
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fi1.rgstatic.net*2Fpublication*2F23485392_Desig
> > n_and_implementation_of_a_sensitive_high-resolution_nonlinear_spectral
> > _imaging_microscope*2Flinks*2F09e4150f7f8c30a99c000000*2Flargepreview.
> > png&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d8ed6
> > d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338766*
> > 7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik
> > 1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=GDf3wIGTkz*2Fe6DUToPUwm*2BgAleM9
> > mgGoSVgUZZwBNEI*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJSU!!C
> > jcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J6X
> > OIb_h$ [nam11[.]safelinks[.]protection[.]outlook[.]com]]<
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
> > dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIj
> > oiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&am
> > p;sdata=1SrT5lAPVXTyVRO2fOXPeyPtVpCGQ2ftpWWZ*2Bn10NTw*3D&reserved=
> > 0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> > (PDF) Design and implementation of a sensitive high-resolution
> > nonlinear spectral imaging microscope<
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fwww.researchgate.net*2Fpublication*2F23485392_
> > Design_and_implementation_of_a_sensitive_high-resolution_nonlinear_spe
> > ctral_imaging_microscope*2Ffigures*3Flo*3D1&data=04*7C01*7Cmmodel*
> > 40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7
> > dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIj
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> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1Jx28NQ6x$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]>
> > 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
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=http*3A*2F*2Fwww.researchgate.net*2F&data=04*7C01*7Cmmod
> > el*40KENT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f7
> > 3e7dd15f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJ
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> > served=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UB
> > tnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J77ThSZ2$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > 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:
> >
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
> > roscopy&data=04*7C01*7Cmmodel*40KENT.EDU*7Ca9523323a2384461a25508d
> > 8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15f26134*7C1*7C0*7C637520402490338
> > 766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTi
> > I6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sdata=SDWAOFXB3UXBeN1uo*2FfFWOsi3Q
> > T2yJ8vUtUyhxXidc8*3D&reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUl!!Cj
> > cC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J7c-
> > lapt$ [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > Post images on
> >
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> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
> > ata=6BMEcMW7H26iSwJyEUipERWLEd6wCNglty8oOMQsxc8*3D&reserved=0__;JS
> > UlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds
> > 6dgWEHngml3WfaZWN7Rk1JzJiy9Uz$
> > [nam11[.]safelinks[.]protection[.]outlook[.]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:
> > >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo> > > ok
> > > .com/?url=http*3A*2F*2Flists__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JwAiKrY_$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7
> > > Cm
> > > model%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d
> > > 01
> > > 8f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3
> > > d8
> > > eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7
> > > C3
> > > 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&r
> > > es
> > > erved=0 Post images on
> > >
> >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=http*3A*2F*2Fwww.imgur.com*2F&data=04*7C01*7Cmmodel*40KE
> > NT.EDU*7Ca9523323a2384461a25508d8ed6d8bd0*7Ce5a06f4a1ec44d018f73e7dd15
> > f26134*7C1*7C0*7C637520402490338766*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC
> > 4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&sd
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> > UlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA26VQtygcy5Kds
> > 6dgWEHngml3WfaZWN7Rk1JzJiy9Uz$
> > [nam11[.]safelinks[.]protection[.]outlook[.]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://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > > nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%
> > > 40
> > > KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7
> > > dd
> > > 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIj
> > > oi
> > > MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&am
> > > p;
> > > sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reser
> > > ve
> > > d=0
> > >
> > > 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://urldefense.com/v3/__https://nam11.safelinks.protection.outlook> > .com/?url=https*3A*2F*2Fwww__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA
> > 26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J80nGEFK$
> > [nam11[.]safelinks[.]protection[.]outlook[.]com].
> > > microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7
> > > C0
> > > 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1
> > > ec
> > > 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpb
> > > GZ
> > > sb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0
> > > %3
> > > D%7C3000&sdata=GCBCeYd7eSP4mi6lYcK3oYOMwiQxZ10b%2F0tVRpIThTA%3D&
> > > am
> > > p;reserved=0 ), 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
> > >
> > >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo> > > ok
> > > .com/?url=http*3A*2F*2Fwww.a__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J-PhdIG6$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > omf.ca%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25
> > > 50
> > > 8d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63752040249
> > > 03
> > > 48759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLC
> > > JB
> > > TiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=FhVEsqwOD9EyXWBTZbMmVwSa
> > > Xs
> > > pqrLCFoREVxEIHTzs%3D&reserved=0<
https://urldefense.com/v3/__https:> > > //nam11.safelinks.protection__;!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBtnpA2
> > > 6V Qtygcy5Kds6dgWEHngml3WfaZWN7Rk1J3rm7zaj$
> > > [nam11[.]safelinks[.]protection]
> > > .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmo
> > > de
> > > l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f
> > > 73
> > > e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8ey
> > > JW
> > > IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C300
> > > 0&
> > > amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserve
> > > d=
> > > 0>
> > >
> > >
> > > -----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/__https://nam11.safelinks.protection.outlo> > > ok
> > > .com/?url=https*3A*2F*2Furld__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > efense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3
> > > Dc
> > > onfocalmicroscopy__%3B!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87
> > > a8
> > > corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm%24&data=04%7C01%7Cmmodel%40KE
> > > NT
> > > .EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15
> > > f2
> > > 6134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC
> > > 4w
> > > LjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sd
> > > at
> > > a=YamE1qZW2OOPk462j1rGa1zJ2O9w8qfDEUdpY2zMbG8%3D&reserved=0
> > > [lists[.]umn[.]edu] Post images on
> > >
https://urldefense.com/v3/__https://nam11.safelinks.protection.outlo> > > ok
> > > .com/?url=https*3A*2F*2Furld__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0UBt
> > > np A26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1JzkdyAOQ$
> > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > efense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!cVq_1Lw
> > > At
> > > t5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60%24&a
> > > mp
> > > ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0
> > > %7
> > > Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnk
> > > no
> > > wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWw
> > > iL
> > > CJXVCI6Mn0%3D%7C3000&sdata=Xkk%2BxZYn%2Fp1oYe%2BcPSDxVVjjyaom5Gp
> > > OF
> > > SVLc6Bztp0%3D&reserved=0 [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.com/v3/__https://nam11.safelinks.protection.out> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cg
> > > > i-
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=355%2FOydVsjZ%2Fyb05O7GUO
> > > > gH
> > > > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > > > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh
> > > > 5o
> > > > fM
> > > > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&
> > > > m=
> > > > aB
> > > > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUD
> > > > Eo
> > > > S-
> > > > 65dOAWbgN2OxNnKaw&e=
> > > > Post images on
> > > >
https://urldefense.com/v3/__https://nam11.safelinks.protection.out> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26
> > > > d%
> > > > 3DDw&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508
> > > > d8
> > > > ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490
> > > > 34
> > > > 8759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiL
> > > > CJ
> > > > BTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gD6IgbED16AsVzP5GvSPq
> > > > 0W
> > > > sRALiXOnn1PZWkjxF9yY%3D&reserved=0
> > > > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGG
> > > > yi
> > > > sI
> > > > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D
> > > > 7C dv 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.com/v3/__https://nam11.safelinks.protection.out> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl
> > > > _-
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN
> > > > %2
> > > > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > > > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0L
> > > > yF
> > > > _z
> > > > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZC
> > > > pE kt
> > > > 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.com/v3/__https://nam11.safelinks.protection.out> > > > lo
> > > > ok.com/?url=https*3A*2F*2Fur__;JSUl!!CjcC7IQ!eEhOfVie8uBJ5Jgg4Ya0U
> > > > Bt npA26VQtygcy5Kds6dgWEHngml3WfaZWN7Rk1J4p9Qajh$
> > > > [nam11[.]safelinks[.]protection[.]outlook[.]com]
> > > > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_
> > > > ar
> > > > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed
> > > > 6d
> > > > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903487
> > > > 59
> > > > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBT
> > > > iI
> > > > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SA
> > > > s%
> > > > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > > > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
> > > > HB
> > > > eT
> > > > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aB
> > > > nP
> > > > uV
> > > > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_C
> > > > yQ
> > > > 63 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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