http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592022.html
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.
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 (
(PDF) Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope<
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
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:
Intensity measured (in micro watt) using 60X (oil) objective Percentage of laser used:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=04%7C01%7Cm
> model%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d01
> 8f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8
> eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3
> 000&sdata=SDWAOFXB3UXBeN1uo%2FfFWOsi3QT2yJ8vUtUyhxXidc8%3D&res
> erved=0 Post images on
>
https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=6BMEcMW7H26iSwJyEUipERWLEd6wCNglty8oOMQsxc8%3D&reserved=0 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> nature.com%2Farticles%2Fs41596-020-0313-9&data=04%7C01%7Cmmodel%40
> KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd
> 15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZsb3d8eyJWIjoi
> MC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> sdata=4Ys8AEy%2FxAfzJCXw%2FclIfrT8GuWUNF9AMZP%2FWZhsgNA%3D&reserve
> 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> microscopyu.com%2Fmicroscopy-basics%2Fimage-brightness&data=04%7C0
> 1%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec
> 44d018f73e7dd15f26134%7C1%7C0%7C637520402490338766%7CUnknown%7CTWFpbGZ
> 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://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.a> omf.ca%2F&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a2550
> 8d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C6375204024903
> 48759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJB
> TiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=FhVEsqwOD9EyXWBTZbMmVwSaXs
> pqrLCFoREVxEIHTzs%3D&reserved=0<
https://nam11.safelinks.protection> .outlook.com/?url=http%3A%2F%2Fwww.aomf.ca%2F&data=04%7C01%7Cmmode
> l%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73
> e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJW
> IjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&
> amp;sdata=FhVEsqwOD9EyXWBTZbMmVwSaXspqrLCFoREVxEIHTzs%3D&reserved=
> 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld> efense.com%2Fv3%2F__http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dc
> onfocalmicroscopy__%3B!!CjcC7IQ!cVq_1LwAtt5kR7u0kLVqLgj6Ibrhi5SENs87a8
> corilw8S_7MAMBm34ZykSs8SUfn_6GBWBm%24&data=04%7C01%7Cmmodel%40KENT
> .EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f2
> 6134%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4w
> LjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdat
> a=YamE1qZW2OOPk462j1rGa1zJ2O9w8qfDEUdpY2zMbG8%3D&reserved=0
> [lists[.]umn[.]edu] Post images on
>
https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Furld> efense.com%2Fv3%2F__http%3A%2F%2Fwww.imgur.com__%3B!!CjcC7IQ!cVq_1LwAt
> t5kR7u0kLVqLgj6Ibrhi5SENs87a8corilw8S_7MAMBm34ZykSs8SUfn8HHnv60%24&
> ;data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d8bd0%7
> Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759%7CUnkno
> wn%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiL
> CJXVCI6Mn0%3D%7C3000&sdata=Xkk%2BxZYn%2Fp1oYe%2BcPSDxVVjjyaom5GpOF
> 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=355%2FOydVsjZ%2Fyb05O7GUOgH
> > xuKsM%2BiK4DjJYGU%2FxrUE%3D&reserved=0
> > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5o
> > fM
> > HBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=
> > aB
> > nPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=NSCBIiLfvnxwocRL4-vTUDEo
> > S-
> > 65dOAWbgN2OxNnKaw&e=
> > Post images on
> >
https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%
> > 3DDw&data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8
> > ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C63752040249034
> > 8759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJ
> > BTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=gD6IgbED16AsVzP5GvSPq0W
> > sRALiXOnn1PZWkjxF9yY%3D&reserved=0
> > IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyi
> > sI
> > eOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpEktGwklB9D7C
> > 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.confocal.nl_-
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=wCEaE8n4FidCGGpODX1yZnEIN%2
> > FYIBPQJgvR7BXli%2FtU%3D&reserved=0
> > 23rcm2&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF
> > _z
> > 8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnPuVl44CvsNnSHKnYuIZtIZCpE
> > 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fur> > ldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__www.nature.com_ar
> > &data=04%7C01%7Cmmodel%40KENT.EDU%7Ca9523323a2384461a25508d8ed6d
> > 8bd0%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C637520402490348759
> > %7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI
> > 6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=3eCTBSNU%2BlS9rvBZUNH66SAs%
> > 2BDLVPItkhqzFMgOpGTM%3D&reserved=0
> > ticles_s41596-2D020-2D0313-2D9&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHB
> > eT
> > l9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=aBnP
> > uV
> > l44CvsNnSHKnYuIZtIZCpEktGwklB9D7Cdvqg&s=WuqudKbziHqalUr5fiK7sSsr_CyQ
> > 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
> >
>
> 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://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
> uhn.ca%2FPatientsFamilies%2FPatient_Safety_Advocacy%2FPrivacy%2FDocume
> nts%2FEmail_consent_and_safety.pdf&data=04%7C01%7Cmmodel%40KENT.ED
> U%7Ca9523323a2384461a25508d8ed6d8bd0%7Ce5a06f4a1ec44d018f73e7dd15f2613
> 4%7C1%7C0%7C637520402490348759%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjA
> wMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=O
> LT4qqVppV23PhWrwzzCwziYyhBG6pcKK5EnOCrurBE%3D&reserved=0>
> 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.
>
CAUTION: EXTERNAL SENDER Do not click any links, open any attachments, or REPLY to the message unless you trust the sender and know the content is safe.