Are lower magnification objectives brighter?

[Nuno Moreno]

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Just adding one more thing:

It is important also the transmittance of the objective. That would depend on the number os lenses inside, glass type, and coating. Of course that all this have an impact at the spectral responses from UV to IR and that is independente of magnification.

Just my 2 cents
\N

> On 26 Mar 2021, at 14:32, Stanislav Vitha <[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.
> *****
>
> Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.
>
> Stan
> Texas A&M University
> Microscopy and Imaging Center
>
> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:
>
>> Hi Arnaud,
>>
>> make sure you have the same
>> power at the sample plane with a power meter
>>
>> You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>> best wishes
>> Andreas
>>

[Arnaud ROYON]

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Hi Andreas and Craig,
Dear list,


@Andreas: you are right, in wide-field microscopy, the irradiance (in
W/cm²) depends on the magnification. My point was about confocal microscopy.

Just to be clear, irradiances for wide-field (WF) and confocal
laser-scanning (CLS) microscopes are given respectively by:

I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
focal plane of the objective, FN the objective field number (mm) and Mag
the objective magnification (unitless).

Note: Sometimes, the field number FN and the field of view FOV can be
mixed-up. They are related through the following equation:FOV=FN⁄Mag.


I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
focal plane of the objective, λ the illumination wavelength (nm) and NA
the objective numerical aperture (unitless).
The factor 4*ln(2) comes from the fact that the spatial distribution of
the laser beam is considered as Gaussian.


@Craig: Yep, I fully agree for the index-matching gel. Though I was not
talking about the coverglass/air and air/sensor interfaces, but about
the 'objective front lens'/'immersion medium' interface.

In figure 23*, from one of the very nice papers Stanley shared with us,
we can see that if we remove the oil, and let air instead, a
non-negligible part of the rays with a high angle will be reflected by
total internal reflection (at the lens/air interface). For this reason,
removing the oil for an oil objective does not indicate if having or not
an index-matching gel between a coverslip and a sensor helps, since part
of the rays will not reach the coverslip anyway.

A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.

*Ref : Y. Zhang and H. Gross, /“Systematic design of microscope
objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
8(5): 313–347.


Best,
Arnaud

Le 26/03/2021 à 15:38, Nuno Moreno a écrit :

> *****
> 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.
> *****
>
> Just adding one more thing:
>
> It is important also the transmittance of the objective. That would depend on the number os lenses inside, glass type, and coating. Of course that all this have an impact at the spectral responses from UV to IR and that is independente of magnification.
>
> Just my 2 cents
> \N
>
>
>
>> On 26 Mar 2021, at 14:32, Stanislav Vitha <[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.
>> *****
>>
>> Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.
>>
>> Stan
>> Texas A&M University
>> Microscopy and Imaging Center
>>
>> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:
>>
>>> Hi Arnaud,
>>>
>>> make sure you have the same
>>> power at the sample plane with a power meter
>>>
>>> You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>>> best wishes
>>> Andreas
>>>

[Benjamin Smith]

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One other thing to keep in mind, especially for laser scanning microscopy
applications, is that if you are overfilling the back aperture you need to
approach the back focal plane at a wider angle to get a 60x FOV with a 60x
objective than to get a 60x FOV with a 20x objective, which means the
average intensity across the full 60x FOV for the 20x objective will be
brighter and sharper even when the NA is the same, due to reduced
vignetting.

To get an intuitive sense of this effect look at the back of an objective,
and compare the cross-sectional area when viewing the back of the objective
straight on vs. off to one side.  As the angle increases, the cross
sectional area of the back aperture decreases, and therefore the average
excitation and emission NA also decreases, resulting in lower resolution
and lower intensity (commonly referred to as vignetting).  Therefore, for
laser scanning applications a 20x/1.0NA objective will be both brighter and
have a higher resolution a 60x/1.0NA objective for the same FOV.  Note,
however, that this difference is pronounced only when the excitation light
is over-filling the back aperture.

You can also confirm this for yourself at the sample plane by oversampling
a 1x FOV (i.e. have the pixel resolution be much higher than the optical
resolution), and then compare the resolution in the center of the FOV vs.
the corners for a 20x/1.0 NA objective at 3x zoom versus a 60x/1.0NA
objective at 1x zoom.  Then, prepare a test slide with a solution of
fluorescein and compare the fluorescence intensity in the middle of the FOV
vs. the corners.

This is why for many in-vivo/ex-vivo laser scanning microscopy imaging
applications, it is best to have just a single high NA/low magnification
objective, as it will always outperform a high magnification objective with
the same numerical aperture.

Cheers,
   Ben Smith

On Fri, Mar 26, 2021 at 8:06 AM Arnaud ROYON <[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 and Craig,
> Dear list,
>
>
> @Andreas: you are right, in wide-field microscopy, the irradiance (in
> W/cm²) depends on the magnification. My point was about confocal
> microscopy.
>
> Just to be clear, irradiances for wide-field (WF) and confocal
> laser-scanning (CLS) microscopes are given respectively by:
>
> I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> focal plane of the objective, FN the objective field number (mm) and Mag
> the objective magnification (unitless).
>
> Note: Sometimes, the field number FN and the field of view FOV can be
> mixed-up. They are related through the following equation:FOV=FN⁄Mag.
>
>
> I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> focal plane of the objective, λ the illumination wavelength (nm) and NA
> the objective numerical aperture (unitless).
> The factor 4*ln(2) comes from the fact that the spatial distribution of
> the laser beam is considered as Gaussian.
>
>
> @Craig: Yep, I fully agree for the index-matching gel. Though I was not
> talking about the coverglass/air and air/sensor interfaces, but about
> the 'objective front lens'/'immersion medium' interface.
>
> In figure 23*, from one of the very nice papers Stanley shared with us,
> we can see that if we remove the oil, and let air instead, a
> non-negligible part of the rays with a high angle will be reflected by
> total internal reflection (at the lens/air interface). For this reason,
> removing the oil for an oil objective does not indicate if having or not
> an index-matching gel between a coverslip and a sensor helps, since part
> of the rays will not reach the coverslip anyway.
>
> A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.
>
> *Ref : Y. Zhang and H. Gross, /“Systematic design of microscope
> objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
> 8(5): 313–347.
>
>
> Best,
> Arnaud
>
> Le 26/03/2021 à 15:38, Nuno Moreno a écrit :
> > *****
> > 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.
> > *****
> >
> > Just adding one more thing:
> >
> > It is important also the transmittance of the objective. That would
> depend on the number os lenses inside, glass type, and coating. Of course
> that all this have an impact at the spectral responses from UV to IR and
> that is independente of magnification.
> >
> > Just my 2 cents
> > \N
> >
> >
> >
> >> On 26 Mar 2021, at 14:32, Stanislav Vitha <[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.
> >> *****
> >>
> >> Assuming the beam is properly expanded, in a point scanning confocal
> system the area illuminated (the Airy disc) is only determined by
> wavelength and the NA of the objective.
> >>
> >> Stan
> >> Texas A&M University
> >> Microscopy and Imaging Center
> >>
> >> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <
> [hidden email]> wrote:
> >>
> >>> Hi Arnaud,
> >>>
> >>> make sure you have the same
> >>> power at the sample plane with a power meter
> >>>
> >>> You also need to take into account the area which is illuminated.
> When you change to lower magnification objectives with the same NA, you
> will illuminate a much larger area, so the power density goes down. This is
> an important factor for the fluorescence intensity, the number of
> excitation photons per fluorophore determines the number of emitted
> photons, if you spread the excitation fluorophores more thinly, you will
> get less fluorescence. If you just measure the LED power through the
> objective, you will miss this point. The question is, if you want to count
> this as "brightness" of the objective or not as you can just change the LED
> power or illumination beam path.
> >>> best wishes
> >>> Andreas
> >>>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Weill Hall
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/

[Christian Wilms]

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*****

Late to the discussion, but once Ben started mentioning in vivo work...

In laser-scanning microscopy without a pinhole (so mainly in multiphoton microscopy), detection is not limited to ballistic photons and that changes things: Once scattered photons are also being detected, the lower the magnification for a given NA, the more light is detected. Warren Zipfel's lab quantified this a few years ago:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541535/

So for multiphoton imaging the drive to use low mag/high NA objectives is of value.

Best, Christian


Dr. Christian Wilms / Research & Development Manager
[hidden email] / [phone]
www.scientifica.uk.com

Take a look at our NeuroWire blog to see our latest news, guides, videos and more.

> -----Original Message-----
> From: Benjamin Smith <[hidden email]>
> Sent: 26 March 2021 19:59
> 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.
> *****
>
> One other thing to keep in mind, especially for laser scanning microscopy
> applications, is that if you are overfilling the back aperture you need to
> approach the back focal plane at a wider angle to get a 60x FOV with a 60x
> objective than to get a 60x FOV with a 20x objective, which means the
> average intensity across the full 60x FOV for the 20x objective will be brighter
> and sharper even when the NA is the same, due to reduced vignetting.
>
> To get an intuitive sense of this effect look at the back of an objective, and
> compare the cross-sectional area when viewing the back of the objective
> straight on vs. off to one side.  As the angle increases, the cross sectional
> area of the back aperture decreases, and therefore the average excitation
> and emission NA also decreases, resulting in lower resolution and lower
> intensity (commonly referred to as vignetting).  Therefore, for laser scanning
> applications a 20x/1.0NA objective will be both brighter and have a higher
> resolution a 60x/1.0NA objective for the same FOV.  Note, however, that this
> difference is pronounced only when the excitation light is over-filling the
> back aperture.
>
> You can also confirm this for yourself at the sample plane by oversampling a
> 1x FOV (i.e. have the pixel resolution be much higher than the optical
> resolution), and then compare the resolution in the center of the FOV vs.
> the corners for a 20x/1.0 NA objective at 3x zoom versus a 60x/1.0NA
> objective at 1x zoom.  Then, prepare a test slide with a solution of fluorescein
> and compare the fluorescence intensity in the middle of the FOV vs. the
> corners.
>
> This is why for many in-vivo/ex-vivo laser scanning microscopy imaging
> applications, it is best to have just a single high NA/low magnification
> objective, as it will always outperform a high magnification objective with the
> same numerical aperture.
>
> Cheers,
>    Ben Smith
>
> On Fri, Mar 26, 2021 at 8:06 AM Arnaud ROYON <[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 and Craig,
> > Dear list,
> >
> >
> > @Andreas: you are right, in wide-field microscopy, the irradiance (in
> > W/cm²) depends on the magnification. My point was about confocal
> > microscopy.
> >
> > Just to be clear, irradiances for wide-field (WF) and confocal
> > laser-scanning (CLS) microscopes are given respectively by:
> >
> > I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))
> >
> > Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> > focal plane of the objective, FN the objective field number (mm) and
> > Mag the objective magnification (unitless).
> >
> > Note: Sometimes, the field number FN and the field of view FOV can be
> > mixed-up. They are related through the following equation:FOV=FN⁄Mag.
> >
> >
> > I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)
> >
> > Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> > focal plane of the objective, λ the illumination wavelength (nm) and
> > NA the objective numerical aperture (unitless).
> > The factor 4*ln(2) comes from the fact that the spatial distribution
> > of the laser beam is considered as Gaussian.
> >
> >
> > @Craig: Yep, I fully agree for the index-matching gel. Though I was
> > not talking about the coverglass/air and air/sensor interfaces, but
> > about the 'objective front lens'/'immersion medium' interface.
> >
> > In figure 23*, from one of the very nice papers Stanley shared with
> > us, we can see that if we remove the oil, and let air instead, a
> > non-negligible part of the rays with a high angle will be reflected by
> > total internal reflection (at the lens/air interface). For this
> > reason, removing the oil for an oil objective does not indicate if
> > having or not an index-matching gel between a coverslip and a sensor
> > helps, since part of the rays will not reach the coverslip anyway.
> >
> > A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.
> >
> > *Ref : Y. Zhang and H. Gross, /“Systematic design of microscope
> > objectives. Part I: System review and analysis,”/ Adv. Opt. Techn.
> > 2019;
> > 8(5): 313–347.
> >
> >
> > Best,
> > Arnaud
> >
> > Le 26/03/2021 à 15:38, Nuno Moreno a écrit :
> > > *****
> > > 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.
> > > *****
> > >
> > > Just adding one more thing:
> > >
> > > It is important also the transmittance of the objective. That would
> > depend on the number os lenses inside, glass type, and coating. Of
> > course that all this have an impact at the spectral responses from UV
> > to IR and that is independente of magnification.
> > >
> > > Just my 2 cents
> > > \N
> > >
> > >
> > >
> > >> On 26 Mar 2021, at 14:32, Stanislav Vitha <[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.
> > >> *****
> > >>
> > >> Assuming the beam is properly expanded, in a point scanning
> > >> confocal
> > system the area illuminated (the Airy disc) is only determined by
> > wavelength and the NA of the objective.
> > >>
> > >> Stan
> > >> Texas A&M University
> > >> Microscopy and Imaging Center
> > >>
> > >> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <
> > [hidden email]> wrote:
> > >>
> > >>> Hi Arnaud,
> > >>>
> > >>> make sure you have the same
> > >>> power at the sample plane with a power meter
> > >>>
> > >>> You also need to take into account the area which is illuminated.
> > When you change to lower magnification objectives with the same NA,
> > you will illuminate a much larger area, so the power density goes
> > down. This is an important factor for the fluorescence intensity, the
> > number of excitation photons per fluorophore determines the number of
> > emitted photons, if you spread the excitation fluorophores more
> > thinly, you will get less fluorescence. If you just measure the LED
> > power through the objective, you will miss this point. The question
> > is, if you want to count this as "brightness" of the objective or not
> > as you can just change the LED power or illumination beam path.
> > >>> best wishes
> > >>> Andreas
> > >>>
> >
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Weill Hall
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-
> imaging/

[Sylvie Le Guyader]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi everyone

Thanks for the interesting discussion. Indeed Arnaud's question has also been puzzling me for a while.
Could one then summarize by saying that, when acquiring images with WF, confocal or multiphoton and with 2 objectives of different magnifications but identical NA, coatings and immersion medium, the image is expected to be brighter with the lowest magnification objective, even if it is for different reasons for the 3 types of microscope?

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arnaud ROYON
Sent: 26 March 2021 16:06
To: [hidden email]
Subject: Re: Are lower magnification objectives brighter?

*****
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Hi Andreas and Craig,
Dear list,


@Andreas: you are right, in wide-field microscopy, the irradiance (in
W/cm²) depends on the magnification. My point was about confocal microscopy.

Just to be clear, irradiances for wide-field (WF) and confocal laser-scanning (CLS) microscopes are given respectively by:

I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the focal plane of the objective, FN the objective field number (mm) and Mag the objective magnification (unitless).

Note: Sometimes, the field number FN and the field of view FOV can be mixed-up. They are related through the following equation:FOV=FN⁄Mag.


I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
focal plane of the objective, λ the illumination wavelength (nm) and NA
the objective numerical aperture (unitless).
The factor 4*ln(2) comes from the fact that the spatial distribution of
the laser beam is considered as Gaussian.


@Craig: Yep, I fully agree for the index-matching gel. Though I was not
talking about the coverglass/air and air/sensor interfaces, but about
the 'objective front lens'/'immersion medium' interface.

In figure 23*, from one of the very nice papers Stanley shared with us,
we can see that if we remove the oil, and let air instead, a
non-negligible part of the rays with a high angle will be reflected by
total internal reflection (at the lens/air interface). For this reason,
removing the oil for an oil objective does not indicate if having or not
an index-matching gel between a coverslip and a sensor helps, since part
of the rays will not reach the coverslip anyway.

A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.

*Ref : Y. Zhang and H. Gross, /“Systematic design of microscope
objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
8(5): 313–347.


Best,
Arnaud

Le 26/03/2021 à 15:38, Nuno Moreno a écrit :

> *****
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> *****
>
> Just adding one more thing:
>
> It is important also the transmittance of the objective. That would depend on the number os lenses inside, glass type, and coating. Of course that all this have an impact at the spectral responses from UV to IR and that is independente of magnification.
>
> Just my 2 cents
> \N
>
>
>
>> On 26 Mar 2021, at 14:32, Stanislav Vitha <[hidden email]> wrote:
>>
>> *****
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>> *****
>>
>> Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.
>>
>> Stan
>> Texas A&M University
>> Microscopy and Imaging Center
>>
>> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:
>>
>>> Hi Arnaud,
>>>
>>> make sure you have the same
>>> power at the sample plane with a power meter
>>>
>>> You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>>> best wishes
>>> Andreas
>>>

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[Jonkman, James]

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Hi, Sylvie.  Who would have thought that it could get so complicated - thanks for trying to summarize what has become a lengthy but interesting discussion!  Personally, I don't like the word "brighter", because it implies that this is the desired outcome.  For laser-scanning confocals, I feel it's misleading to tell a user that a lower mag objective gives you a brighter image, when it's definitely only "brighter" in the sense that you're hitting it with more excitation light so it is not an advantage at all.  With CLSM I prefer to avoid telling people that lower mag = brighter, and instead emphasize that higher NA = more efficient light collection (2x NA gives you 4x more light collection).

There have been many other interesting comments on this thread.  I liked Andreas's widefield measurements.  I hadn't thought of binning to get the same pixel size.  The old formulae that originally inspired people to think that 'lower mag = brighter' were of course developed assuming that you would observe the sample in the binocular; and you can't bin your photoreceptors, so what's the most fair comparison?  As you wish I suppose!

Interesting comment about two-photon as well.  We primarily use a 20x/1.0NA water immersion lens for intravital 2p imaging.  I had not really considered the light-collection advantage that the lower mag offers for Non-descanned detection - glad you pointed this out, Christian (and nice paper, Zipfel lab!).

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: [phone]
   www.aomf.ca


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
Sent: Monday, March 29, 2021 8:31 AM
To: [hidden email]
Subject: [External] Re: Are lower magnification objectives brighter?

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Hi everyone

Thanks for the interesting discussion. Indeed Arnaud's question has also been puzzling me for a while.
Could one then summarize by saying that, when acquiring images with WF, confocal or multiphoton and with 2 objectives of different magnifications but identical NA, coatings and immersion medium, the image is expected to be brighter with the lowest magnification objective, even if it is for different reasons for the 3 types of microscope?

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
Follow our microscopy blog!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arnaud ROYON
Sent: 26 March 2021 16:06
To: [hidden email]
Subject: Re: Are lower magnification objectives brighter?

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*****

Hi Andreas and Craig,
Dear list,


@Andreas: you are right, in wide-field microscopy, the irradiance (in
W/cm²) depends on the magnification. My point was about confocal microscopy.

Just to be clear, irradiances for wide-field (WF) and confocal laser-scanning (CLS) microscopes are given respectively by:

I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the focal plane of the objective, FN the objective field number (mm) and Mag the objective magnification (unitless).

Note: Sometimes, the field number FN and the field of view FOV can be mixed-up. They are related through the following equation:FOV=FN⁄Mag.


I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)

Where I is the irradiance (W.cm-2), Pav the average power (mW) at the focal plane of the objective, λ the illumination wavelength (nm) and NA the objective numerical aperture (unitless).
The factor 4*ln(2) comes from the fact that the spatial distribution of the laser beam is considered as Gaussian.


@Craig: Yep, I fully agree for the index-matching gel. Though I was not talking about the coverglass/air and air/sensor interfaces, but about the 'objective front lens'/'immersion medium' interface.

In figure 23*, from one of the very nice papers Stanley shared with us, we can see that if we remove the oil, and let air instead, a non-negligible part of the rays with a high angle will be reflected by total internal reflection (at the lens/air interface). For this reason, removing the oil for an oil objective does not indicate if having or not an index-matching gel between a coverslip and a sensor helps, since part of the rays will not reach the coverslip anyway.

A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.

*Ref : Y. Zhang and H. Gross, /“Systematic design of microscope objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
8(5): 313–347.


Best,
Arnaud

Le 26/03/2021 à 15:38, Nuno Moreno a écrit :

> *****
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> *****
>
> Just adding one more thing:
>
> It is important also the transmittance of the objective. That would depend on the number os lenses inside, glass type, and coating. Of course that all this have an impact at the spectral responses from UV to IR and that is independente of magnification.
>
> Just my 2 cents
> \N
>
>
>
>> On 26 Mar 2021, at 14:32, Stanislav Vitha <[hidden email]> wrote:
>>
>> *****
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>> *****
>>
>> Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.
>>
>> Stan
>> Texas A&M University
>> Microscopy and Imaging Center
>>
>> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:
>>
>>> Hi Arnaud,
>>>
>>> make sure you have the same
>>> power at the sample plane with a power meter
>>>
>>> You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>>> best wishes
>>> Andreas
>>>

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On Mon, Mar 29, 2021 at 8:21 AM Jonkman, James <[hidden email]>
wrote:

> Hi, Sylvie.  [...]
> Interesting comment about two-photon as well.  We primarily use a
> 20x/1.0NA water immersion lens for intravital 2p imaging.  I had not really
> considered the light-collection advantage that the lower mag offers for
> Non-descanned detection - glad you pointed this out, Christian (and nice
> paper, Zipfel lab!).
>
> For 2P any photon is a valid signal photon because the excitation
conditions specify that any light generated must have come from the focal
volume of the laser. As long as your laser is correctly focused then you
are getting your diffraction-limited near NA-based spot size on the sample.
(scattering, index changes etc thwart this somewhat but that's another
discussion)
So if any photon is valid signal, you want to have as large a chance of
catching it as you possibly can. First the NA of the objective helps by
increasing the acceptance angle over which stray photons will be captured,
but it didn't occur to me until this discussion that a lower mag objective
will have a larger field of view, which also improves the chance of
catching a stray scattered photon. In the limit, the highest NA lowest mag
lens you can get is best for 2P.

I'm thinking a bit to see how this works out with confocal as the pinhole
is your limiting factor in that case. Assuming the pinhole is matched to
the airy disk of the laser spot or system aperture then only NA should
matter, I think, as the pinhole restricts light to the aperture airy disk
projected on to the sample.

Craig

>
>
> -----------------------------------------------
>    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: [phone]
>    www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Sylvie Le Guyader
> Sent: Monday, March 29, 2021 8:31 AM
> To: [hidden email]
> Subject: [External] Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> [imgur[.]com] and include the link in your posting.
> *****
>
> Hi everyone
>
> Thanks for the interesting discussion. Indeed Arnaud's question has also
> been puzzling me for a while.
> Could one then summarize by saying that, when acquiring images with WF,
> confocal or multiphoton and with 2 objectives of different magnifications
> but identical NA, coatings and immersion medium, the image is expected to
> be brighter with the lowest magnification objective, even if it is for
> different reasons for the 3 types of microscope?
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Arnaud ROYON
> Sent: 26 March 2021 16:06
> To: [hidden email]
> Subject: Re: Are lower magnification objectives brighter?
>
> *****
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> [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in
> your posting.
> *****
>
> Hi Andreas and Craig,
> Dear list,
>
>
> @Andreas: you are right, in wide-field microscopy, the irradiance (in
> W/cm²) depends on the magnification. My point was about confocal
> microscopy.
>
> Just to be clear, irradiances for wide-field (WF) and confocal
> laser-scanning (CLS) microscopes are given respectively by:
>
> I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> focal plane of the objective, FN the objective field number (mm) and Mag
> the objective magnification (unitless).
>
> Note: Sometimes, the field number FN and the field of view FOV can be
> mixed-up. They are related through the following equation:FOV=FN⁄Mag.
>
>
> I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the
> focal plane of the objective, λ the illumination wavelength (nm) and NA the
> objective numerical aperture (unitless).
> The factor 4*ln(2) comes from the fact that the spatial distribution of
> the laser beam is considered as Gaussian.
>
>
> @Craig: Yep, I fully agree for the index-matching gel. Though I was not
> talking about the coverglass/air and air/sensor interfaces, but about the
> 'objective front lens'/'immersion medium' interface.
>
> In figure 23*, from one of the very nice papers Stanley shared with us, we
> can see that if we remove the oil, and let air instead, a non-negligible
> part of the rays with a high angle will be reflected by total internal
> reflection (at the lens/air interface). For this reason, removing the oil
> for an oil objective does not indicate if having or not an index-matching
> gel between a coverslip and a sensor helps, since part of the rays will not
> reach the coverslip anyway.
>
> A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.
>
> *Ref : Y. Zhang and H. Gross, /“Systematic design of microscope
> objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
> 8(5): 313–347.
>
>
> Best,
> Arnaud
>
> Le 26/03/2021 à 15:38, Nuno Moreno a écrit :
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook
> > .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
> > roscopy&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c
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> [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in
> your posting.
> > *****
> >
> > Just adding one more thing:
> >
> > It is important also the transmittance of the objective. That would
> depend on the number os lenses inside, glass type, and coating. Of course
> that all this have an impact at the spectral responses from UV to IR and
> that is independente of magnification.
> >
> > Just my 2 cents
> > \N
> >
> >
> >
> >> On 26 Mar 2021, at 14:32, Stanislav Vitha <[hidden email]> wrote:
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> https://urldefense.com/v3/__https://eur01.safelinks.protection.outloo
> >> k.com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalm
> >> icroscopy&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a34
> >> 41c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637
> >> 523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV
> >> 2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=Zqp1OYp0paVR9F
> >> cTsJq7HOOZN*2Bc1mx4ESwbLB*2BQrZjg*3D&amp;reserved=0__;JSUlJSUlJSUlJSU
> >> lJSUlJSUlJSUlJQ!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiM
> >> UxfKIz1eAk8s0nrAiiSLm2U_$
> >> [eur01[.]safelinks[.]protection[.]outlook[.]com]
> >> Post images on
> https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=UREzKWYKm8ym3s*2B8T4bxGa6TsepCcCgWDX0wiY*2FA*2FXs*3D&amp;reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAiknfWuIB$
> [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in
> your posting.
> >> *****
> >>
> >> Assuming the beam is properly expanded, in a point scanning confocal
> system the area illuminated (the Airy disc) is only determined by
> wavelength and the NA of the objective.
> >>
> >> Stan
> >> Texas A&M University
> >> Microscopy and Imaging Center
> >>
> >> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <
> [hidden email]> wrote:
> >>
> >>> Hi Arnaud,
> >>>
> >>> make sure you have the same
> >>> power at the sample plane with a power meter
> >>>
> >>> You also need to take into account the area which is illuminated.
> When you change to lower magnification objectives with the same NA, you
> will illuminate a much larger area, so the power density goes down. This is
> an important factor for the fluorescence intensity, the number of
> excitation photons per fluorophore determines the number of emitted
> photons, if you spread the excitation fluorophores more thinly, you will
> get less fluorescence. If you just measure the LED power through the
> objective, you will miss this point. The question is, if you want to count
> this as "brightness" of the objective or not as you can just change the LED
> power or illumination beam path.
> >>> best wishes
> >>> Andreas
> >>>
>
>
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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 http://www.imgur.com and include the link in your posting.
*****

Hi James,

Binoculars: Some of the microscope reps used to put 18mm eyepieces on
their demo microscopes when competing for brightest fluorescence by eye.
Whether they were encouraged to do this by their manager (or their CEO,
if the CEO knew anything about microscopes and/or selling), I do not
know. An alternative, or complementary hypothesis, is that some
salespeople learned from inspecting their competitors microscopes. Of
course splitting the light to two eyepieces is cutting down the light
that could have gone n to a monocular port eyepiece.

I wrote this several years ago
(https://works.bepress.com/gmcnamara/85/     -- ahhh, please save a few
trees by NOT hitting the print button):

Your Sales Representative is As Important As The Company

You may think you are buying a Leica, Nikon, Olympus or Zeiss
microscope, but in reality you are buying a piece of your local sales
representative. Most sales representatives move from company to company
and/or territory to territory several times in their career (and if you
don’t believe this, ask them!). For example, the best sales rep I ever
had was previously a shoe salesman (his initials are KN, and since I met
him he moved from a dealership to a microscope company).

The best sales representatives understand your applications, listen to
your needs and wants, maximize the amount of your budget that ends up in
the purchase order, deliver all items within a reasonable time, and
provide training and service.  With respect to “needs vs. wants” I refer
you to the words of the Rolling Stones’ song, “You can’t always get what
you want…”. If you nickel and dime your salesperson, you should not
expect great service from them.

The worst salespeople are the ones who lie about their products and
their competitor’s products, and who cheat during demo’s. A classic
trick, formerly (1980’s, hopefully not practiced today) was that brand
“[2021 update: likely all four companies had reps doing this - and some
may still]” reps would demo fluorescence microscope systems with a
smaller diameter eyepiece than their competitor’s (i.e. 18 vs. 22 mm,
back then, 22 vs. 25 or 28 mm now). Sending more light through a smaller
eyepiece gives the user the impression of a brighter specimen. This is
why I counsel that you should use your digital camera to quantify image
brightness in demo’s. Unfortunately, this is probably not a feasible
method unless you already have a complete system (camera, software and
microscope) and are simply looking to add a new microscope. This is
because the reps could still cheat by demo’ing with different
fluorescence filter sets and different light sources than what you are
being quoted. I welcome email from salespeople about other tricks and
will respect their anonymity if they so desire.


***

I also note that our eyes take time to dark adapt (~15 minutes to see
the colors of stars, clear night, no city glare, etc) AND microscope
rooms are usually not dark enough to fully dark adapt (hint: PC monitor).

I am happy to see there are know a lot more than four microscope
companies to choose from -- best wishes to all honest salespeople and
customers.

happy 2021,

George

On 3/29/2021 10:21 AM, Jonkman, James 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, Sylvie.  Who would have thought that it could get so complicated - thanks for trying to summarize what has become a lengthy but interesting discussion!  Personally, I don't like the word "brighter", because it implies that this is the desired outcome.  For laser-scanning confocals, I feel it's misleading to tell a user that a lower mag objective gives you a brighter image, when it's definitely only "brighter" in the sense that you're hitting it with more excitation light so it is not an advantage at all.  With CLSM I prefer to avoid telling people that lower mag = brighter, and instead emphasize that higher NA = more efficient light collection (2x NA gives you 4x more light collection).
>
> There have been many other interesting comments on this thread.  I liked Andreas's widefield measurements.  I hadn't thought of binning to get the same pixel size.  The old formulae that originally inspired people to think that 'lower mag = brighter' were of course developed assuming that you would observe the sample in the binocular; and you can't bin your photoreceptors, so what's the most fair comparison?  As you wish I suppose!
>
> Interesting comment about two-photon as well.  We primarily use a 20x/1.0NA water immersion lens for intravital 2p imaging.  I had not really considered the light-collection advantage that the lower mag offers for Non-descanned detection - glad you pointed this out, Christian (and nice paper, Zipfel lab!).
>
> 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: [phone]
>     www.aomf.ca
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader
> Sent: Monday, March 29, 2021 8:31 AM
> To: [hidden email]
> Subject: [External] 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!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAig6v3xel$ [lists[.]umn[.]edu] Post images on https://urldefense.com/v3/__http://www.imgur.com__;!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAit5eW48f$ [imgur[.]com] and include the link in your posting.
> *****
>
> Hi everyone
>
> Thanks for the interesting discussion. Indeed Arnaud's question has also been puzzling me for a while.
> Could one then summarize by saying that, when acquiring images with WF, confocal or multiphoton and with 2 objectives of different magnifications but identical NA, coatings and immersion medium, the image is expected to be brighter with the lowest magnification objective, even if it is for different reasons for the 3 types of microscope?
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader, PhD
> Live Cell Imaging Facility Manager
> Karolinska Institutet- Bionut Dpt
> Blickagången 16,
> Room 7362 (lab)/7840 (office)
> 14157 Huddinge, Sweden
> mobile: [phone]
> LCI website
> Follow our microscopy blog!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Arnaud ROYON
> Sent: 26 March 2021 16:06
> To: [hidden email]
> Subject: Re: Are lower magnification objectives brighter?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook.com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmicroscopy&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=Zqp1OYp0paVR9FcTsJq7HOOZN*2Bc1mx4ESwbLB*2BQrZjg*3D&amp;reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSUlJQ!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAiiSLm2U_$ [eur01[.]safelinks[.]protection[.]outlook[.]com]
> Post images on https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=UREzKWYKm8ym3s*2B8T4bxGa6TsepCcCgWDX0wiY*2FA*2FXs*3D&amp;reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAiknfWuIB$ [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in your posting.
> *****
>
> Hi Andreas and Craig,
> Dear list,
>
>
> @Andreas: you are right, in wide-field microscopy, the irradiance (in
> W/cm²) depends on the magnification. My point was about confocal microscopy.
>
> Just to be clear, irradiances for wide-field (WF) and confocal laser-scanning (CLS) microscopes are given respectively by:
>
> I WF = (0.1 * Pav) / (PI * (FN / (2 * Mag)^2))
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the focal plane of the objective, FN the objective field number (mm) and Mag the objective magnification (unitless).
>
> Note: Sometimes, the field number FN and the field of view FOV can be mixed-up. They are related through the following equation:FOV=FN⁄Mag.
>
>
> I CLS = (10^11 * 4 * ln(2) * Pav) / (PI * (0.52 * λ / NA)^2)
>
> Where I is the irradiance (W.cm-2), Pav the average power (mW) at the focal plane of the objective, λ the illumination wavelength (nm) and NA the objective numerical aperture (unitless).
> The factor 4*ln(2) comes from the fact that the spatial distribution of the laser beam is considered as Gaussian.
>
>
> @Craig: Yep, I fully agree for the index-matching gel. Though I was not talking about the coverglass/air and air/sensor interfaces, but about the 'objective front lens'/'immersion medium' interface.
>
> In figure 23*, from one of the very nice papers Stanley shared with us, we can see that if we remove the oil, and let air instead, a non-negligible part of the rays with a high angle will be reflected by total internal reflection (at the lens/air interface). For this reason, removing the oil for an oil objective does not indicate if having or not an index-matching gel between a coverslip and a sensor helps, since part of the rays will not reach the coverslip anyway.
>
> A 1.4 NA oil objective with oil becomes a 0.65 NA objective without.
>
> *Ref : Y. Zhang and H. Gross, /“Systematic design of microscope objectives. Part I: System review and analysis,”/ Adv. Opt. Techn. 2019;
> 8(5): 313–347.
>
>
> Best,
> Arnaud
>
> Le 26/03/2021 à 15:38, Nuno Moreno a écrit :
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook
>> .com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalmic
>> roscopy&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c
>> 83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C6375236
>> 79963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMz
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>> UlJSUlJQ!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1e
>> Ak8s0nrAiiSLm2U_$ [eur01[.]safelinks[.]protection[.]outlook[.]com]
>> Post images on https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=UREzKWYKm8ym3s*2B8T4bxGa6TsepCcCgWDX0wiY*2FA*2FXs*3D&amp;reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAiknfWuIB$ [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in your posting.
>> *****
>>
>> Just adding one more thing:
>>
>> It is important also the transmittance of the objective. That would depend on the number os lenses inside, glass type, and coating. Of course that all this have an impact at the spectral responses from UV to IR and that is independente of magnification.
>>
>> Just my 2 cents
>> \N
>>
>>
>>
>>> On 26 Mar 2021, at 14:32, Stanislav Vitha <[hidden email]> wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
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>>> k.com/?url=http*3A*2F*2Flists.umn.edu*2Fcgi-bin*2Fwa*3FA0*3Dconfocalm
>>> icroscopy&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a34
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>>> Post images on https://urldefense.com/v3/__https://eur01.safelinks.protection.outlook.com/?url=http*3A*2F*2Fwww.imgur.com*2F&amp;data=04*7C01*7Csylvie.le.guyader*40KI.SE*7C24ae310e8a3441c83ed908d8f068be43*7Cbff7eef1cf4b4f32be3da1dda043c05d*7C0*7C0*7C637523679963485696*7CUnknown*7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0*3D*7C3000&amp;sdata=UREzKWYKm8ym3s*2B8T4bxGa6TsepCcCgWDX0wiY*2FA*2FXs*3D&amp;reserved=0__;JSUlJSUlJSUlJSUlJSUlJSUlJSU!!CjcC7IQ!aCqT-6r8tlHeC0dC7kAhBaerkboyMb5aKLIUft4-6wiMUxfKIz1eAk8s0nrAiknfWuIB$ [eur01[.]safelinks[.]protection[.]outlook[.]com] and include the link in your posting.
>>> *****
>>>
>>> Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.
>>>
>>> Stan
>>> Texas A&M University
>>> Microscopy and Imaging Center
>>>
>>> On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:
>>>
>>>> Hi Arnaud,
>>>>
>>>> make sure you have the same
>>>> power at the sample plane with a power meter
>>>>
>>>> You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path.
>>>> best wishes
>>>> Andreas
>>>>
>
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