http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592035.html
are reflected. This artificially decreases the NA of the objective.
Select an oil objective, and image the sample with and without oil.
Cité de la Photonique, Bat. Elnath
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> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
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>
> Another subtlety when considering camera binning: combining 4 pixels to
> make one larger effective pixel loses a bit of light due to the dead space
> "+" between the four pixels. CMOS and CCD also handle binning differently
> which is something to consider.
>
> Regarding power throughput for objectives: For some higher-end laser
> scanning systems there is a variable telescope built into the scanhead on
> the excitation input. Our Nikon A1 had this feature for 2P mode. When you
> switched lenses it would move the telescope elements and change the beam
> diameter to better match the back aperture of the current objective. For
> lower mag lenses it would widen the beam, and for higher mag lenses it
> would shrink the beam. Simpler systems use a fixed wide beam diameter to
> match the worst-case low mag scenario and just eat the power loss when
> using a higher mag smaller back aperture lens assuming the NA increase will
> help make up the difference.
>
> Craig
>
> On Wed, Mar 24, 2021 at 1:30 PM Andreas Bruckbauer <
>
[hidden email]> wrote:
>
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>>
>> 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:
>>
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>>
>> 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:
>>
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>> 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:
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>>>
>>
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:
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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
>>>>
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