Power/cm2

[Carmen Sánchez]

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Dear all,
we need to measure the power of light/cm2 in a widefield microscope. We usually use a Newport Power Meter 842-PE to monitor LEDs and laser potencies in our microscopes but… how can we divide mW by the area?
Thanks in advance
Kind regards
Carmen

[Craig Brideau]

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You can estimate based on the size of your field of view. Otherwise, you
have to put a piece of paper at the focal plane and try to estimate the
spot size. I've actually projected the spot from an objective on to a ruler
and measured it that way, but it was for a very low-mag lens so the spot
was pretty big. I've also used calipers and narrowed them until I observed
beam clipping. These are all pretty crude methods but should get you in the
ballpark. Sometimes you just have to use what you have on hand.

Craig

On Thu, Aug 27, 2020 at 9:04 AM Carmen Sánchez <[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.
> *****
>
> Dear all,
> we need to measure the power of light/cm2 in a widefield microscope. We
> usually use a Newport Power Meter 842-PE to monitor LEDs and laser
> potencies in our microscopes but… how can we divide mW by the area?
> Thanks in advance
> Kind regards
> Carmen
>

[George Patterson]

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Hi,
An alternative which we've used is to photobleach a homogeneous pool of
immobilized fluorophore.
I've not found this approach discussed elsewhere, so this might be a good
opportunity for feedback on potential pitfalls that I'm not considering.
We immobilize fluorophore in polyacrylamide polymerized between two
coverslips. We typically have used purified fluorescent protein but I don't
why see a fluorescent dextran or fluorescent BSA won't work.
Image the same field continuously to photobleach the fluorophore. Usually,
I do many of these spaced a field of view or two apart. It makes them
easier to find in the next step.
Switch to a lower magnification objective lens and find one or more of the
photobleached spots. If your microscope is calibrated for that objective
lens, you should be able to get a measure of the excitation spot size and
shape. If it's not calibrated, an appropriate graticule can help with that.
If you want to do this for all objectives, it obviously won't work for the
lowest mag objective. However, one of the approaches discussed earlier
should work.
I do realize that not everyone commonly has polyacrylamide in solution
since many rely on precast gels. Agarose might be an alternative, but make
sure the probe can't diffuse too much while you are photobleaching.
Probably the easiest would be to dry some fluorophore on a coverslip. Even
after adding buffer for imaging, there will likely be a lot still adhered
to the coverslip. You could probably just use a microliter of fluorescent
secondary antibody. It likely will not be a pretty image, but should be
good enough to determine the spot size.
Best regards,
George






On Thu, Aug 27, 2020 at 11:16 AM Craig Brideau <[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.
> *****
>
> You can estimate based on the size of your field of view. Otherwise, you
> have to put a piece of paper at the focal plane and try to estimate the
> spot size. I've actually projected the spot from an objective on to a ruler
> and measured it that way, but it was for a very low-mag lens so the spot
> was pretty big. I've also used calipers and narrowed them until I observed
> beam clipping. These are all pretty crude methods but should get you in the
> ballpark. Sometimes you just have to use what you have on hand.
>
> Craig
>
> On Thu, Aug 27, 2020 at 9:04 AM Carmen Sánchez <[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.
> > *****
> >
> > Dear all,
> > we need to measure the power of light/cm2 in a widefield microscope. We
> > usually use a Newport Power Meter 842-PE to monitor LEDs and laser
> > potencies in our microscopes but… how can we divide mW by the area?
> > Thanks in advance
> > Kind regards
> > Carmen
> >
>

[Zdenek Svindrych-2]

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Hi George,

yes, this is a perfectly valid way to do this if you don't have an
adjustable field stop in your fluorescence illumination path.
One pitfall I can see is that the boundary of the bleached region may not
be sharp, either due to diffusion of the dye (depending how you prepare
your polyacrylamide, the dye may not be immobilised, that's the principle
of PAGE after all), or due to poorly defined illuminated region (no sharp
field stop in the illumination path; note that objective lenses do not have
field stop inside, only the aperture stop).
It's trivial to calculate the power/cm^2 if the illumination is
homogeneous. If not, it's possible to estimate the illumination profile
from the photobleached spots, I think it has been published somewhere, I
just can't remember the title of that paper...

And I'll repeat what I've said several times already, if your illumination
setup has both variable field stop and variable aperture stop, it's trivial
to measure the illumination intensity (W/cm^2) using a regular power meter
and a low-mag objective lens: With your lens of interest, close the field
stop a bit so you can see it on the camera and measure the illuminated
area. Close down the aperture stop so it just barely clips any light that
would pass through the objective lens's back focal plane (BFP) aperture (a
Bertrand lens or an eyepiece telescope is really useful to make sure the
aperture stop is centered and sized properly). Then switch to a low mag dry
lens (make sure it's BFP aperture is larger than that of the lens of
interest; or if you have a large active area detector, you can put it in
place of the objective lens) and measure the power. If you want a more
accurate result, you can factor in the transmission curves of the lenses
and the angular dependence of the detector, but these effects won't be huge.

Best, zdenek


On Sat, Aug 29, 2020 at 1:58 PM George Patterson <[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,
> An alternative which we've used is to photobleach a homogeneous pool of
> immobilized fluorophore.
> I've not found this approach discussed elsewhere, so this might be a good
> opportunity for feedback on potential pitfalls that I'm not considering.
> We immobilize fluorophore in polyacrylamide polymerized between two
> coverslips. We typically have used purified fluorescent protein but I don't
> why see a fluorescent dextran or fluorescent BSA won't work.
> Image the same field continuously to photobleach the fluorophore. Usually,
> I do many of these spaced a field of view or two apart. It makes them
> easier to find in the next step.
> Switch to a lower magnification objective lens and find one or more of the
> photobleached spots. If your microscope is calibrated for that objective
> lens, you should be able to get a measure of the excitation spot size and
> shape. If it's not calibrated, an appropriate graticule can help with that.
> If you want to do this for all objectives, it obviously won't work for the
> lowest mag objective. However, one of the approaches discussed earlier
> should work.
> I do realize that not everyone commonly has polyacrylamide in solution
> since many rely on precast gels. Agarose might be an alternative, but make
> sure the probe can't diffuse too much while you are photobleaching.
> Probably the easiest would be to dry some fluorophore on a coverslip. Even
> after adding buffer for imaging, there will likely be a lot still adhered
> to the coverslip. You could probably just use a microliter of fluorescent
> secondary antibody. It likely will not be a pretty image, but should be
> good enough to determine the spot size.
> Best regards,
> George
>
>
>
>
>
>
> On Thu, Aug 27, 2020 at 11:16 AM Craig Brideau <[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.
> > *****
> >
> > You can estimate based on the size of your field of view. Otherwise, you
> > have to put a piece of paper at the focal plane and try to estimate the
> > spot size. I've actually projected the spot from an objective on to a
> ruler
> > and measured it that way, but it was for a very low-mag lens so the spot
> > was pretty big. I've also used calipers and narrowed them until I
> observed
> > beam clipping. These are all pretty crude methods but should get you in
> the
> > ballpark. Sometimes you just have to use what you have on hand.
> >
> > Craig
> >
> > On Thu, Aug 27, 2020 at 9:04 AM Carmen Sánchez <[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.
> > > *****
> > >
> > > Dear all,
> > > we need to measure the power of light/cm2 in a widefield microscope. We
> > > usually use a Newport Power Meter 842-PE to monitor LEDs and laser
> > > potencies in our microscopes but… how can we divide mW by the area?
> > > Thanks in advance
> > > Kind regards
> > > Carmen
> > >
> >
>

--
--
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Petro]

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If you have motorized stage you can use edge method to get size of the spot.
Best.
Petro.

On Thu, Aug 27, 2020, 5:04 PM Carmen Sánchez <[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.
> *****
>
> Dear all,
> we need to measure the power of light/cm2 in a widefield microscope. We
> usually use a Newport Power Meter 842-PE to monitor LEDs and laser
> potencies in our microscopes but… how can we divide mW by the area?
> Thanks in advance
> Kind regards
> Carmen
>

[Arnaud ROYON]

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Dear Carmen,

One year and a half ago, Argolight released an all-in-one product having
a slide format, the Argo-POWER-HM
(http://argolight.com/products/argo-power/), combining an optical power
meter and a glass containing fluorescent patterns, dedicated to assess
several performance aspects of fluorescence microscopes.

The product comes with a software, Daybook
(http://argolight.com/measure-microscopes-performances-detect-issues-with-daybook/),
that for instance allows to read the optical power (in W) measured with
the power meter at the sample location, and can provide directly an
estimation of the irradiance (in W/cm²), for both wide-field and
confocal laser-scanning microscopes, based on theoretical calculations
of the beam area.

The formulas used in Daybook can be found in the user guide of the
Argo-POWER-HM product, page 31:
http://argolight.com/files/Argo-POWER/userguide/Userguide-Argo-POWER-HM.pdf
Providing you measure the optical power at the sample location (/i.e./
around the focal plane of the objective), you can use the formula suited
to a wide-field illumination for your application.

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: [hidden email]
Tel: [phone]
Web site: www.argolight.com

Le 27/08/2020 à 16:50, Carmen S ánchez 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.
> *****
>
> Dear all,
> we need to measure the power of light/cm2 in a widefield microscope. We usually use a Newport Power Meter 842-PE to monitor LEDs and laser potencies in our microscopes but… how can we divide mW by the area?
> Thanks in advance
> Kind regards
> Carmen