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Dear list,

/"You can test this yourself by measuring a high NA oil lens with and 
without oil on the sensor."/

This test might give you some indications, but I do not think it would 
prove that all (or most of) the rays are collected by a power sensor 
containing an index-matching gel.
In confocal microscopy, when using an oil objective without oil on the 
sensor, there is another interface to consider, where total internal 
reflection (TIR) occurs: it is the interface between the objective front 
lens and the immersion medium (/i.e./ air if no oil is used).
At this interface, the ray with angles higher than the TIR limit angle 
are reflected. This artificially decreases the NA of the objective.

Maybe a more suitable test would be to measure the power coming out of 
the oil objective (with oil on the sensor) with a power meter containing 
an index-matching gel, and the same power meter with the index-matching 
gel removed.

However, going back to the initial topic of this discussion, but from 
the NA perspective, it is actually a good way to check how the 
brightness evolves with NA.
Take a stable fluorescent sample, ideally just below a cover-slip (to be 
in the design configuration of the cover-slip corrected objectives).
Select an oil objective, and image the sample with and without oil. 
Magnification remains the same, while numerical aperture changes.
You will directly notice a drop of contrast (due to degraded resolution) 
and brightness.
In wide-field microscopy, this is due to TIR at the coverslip / air 
interface (rays are coming from the sample to the objective).
In confocal microscopy, this is due to TIR at the objective front lens / 
air interface (rays are coming from the objective to the sample).
If you can estimate (from Snell-Descartes calculations for instance) the 
objective NA without oil, you can verify the formula "Brightness=NA^4 / 
Mag^2 ".
Or, if you can measure the drop of brightness, you can determine the 
drop of NA when no oil is put on the coverslip.

 From the magnification perspective, as James wrote, take two objectives 
with different magnifications and same NA, make sure you have the same 
power at the sample plane with a power meter (you do not necessarily 
need to measure the absolute values, since you are comparing two 
powers), image a stable sample, and check how the brightness evolves 
with magnification.

Best regards,
Arnaud

Arnaud ROYON, Ph.D.
CSO & CTO, Member of the Executive Board, Co-founder
Argolight
Cité de la Photonique, Bat. Elnath
11 avenue de Canteranne
33600 Pessac, FRANCE
Email: [log in to unmask]
Tel: (+33) 5 64 31 08 50
Web site: www.argolight.com

Le 24/03/2021 à 21:12, Craig Brideau a écrit :
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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 <
> [log in to unmask]> 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 <[log in to unmask]>
>> To: [log in to unmask]
>> 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 <
>> [log in to unmask]> 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
>>>   [log in to unmask]  Tel: 416-581-8593
>>>     www.aomf.ca
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[log in to unmask]]
>>> On Behalf Of Michael Giacomelli
>>> Sent: Monday, March 22, 2021 1:10 PM
>>> To: [log in to unmask]
>>> 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 <
>>> [log in to unmask]> wrote:
>>>
>>>> *****
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>>>> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=kbmfwr1Yojg42sGEpaQh5ofM
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>>>> 65dOAWbgN2OxNnKaw&e=
>>>> Post images on
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>>>> IFaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisI
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>>>> 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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