Posted by
Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Assessing-phototoxicity-in-live-fluorescence-imaging-tp7587005p7587015.html
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Hi Steffen,
you can improve the accuracy of the method "a)" (that is measuring the laser
power before reaching the objective) greatly by mounting an iris stop in
place of the lens, adjusting the aperture to equal the diameter of the back
focal plane (BFP) aperture of the objective lens, and measuring the power of
the light that gets through this aperture.
Most confocal microscopes 'overfill' the BFP greatly (which is good for
resolution) and you can get an order of magnitude difference when the laser
beam is > 15 mm 'diameter' (it's gaussian profile) and the BFP diameter is
just 4 mm (e.g. some high-magnification lenses).
You can determine the BFP diameter by looking at the back of the lens, or by
dong some simple math (I guess diameter = 2 * NA * focal_length; focal_
length = tube_lens_focal_lenght / magnification).
This way, your a) and b) results should be much closer to each other and to
the real value in between...
Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/tel: 434-982-4869
---------- Původní e-mail ----------
Od: Steffen Dietzel <
[hidden email]>
Komu:
[hidden email]
Datum: 19. 7. 2017 7:03:20
Předmět: Re: Assessing phototoxicity in live fluorescence imaging
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Unfortunately not a solution, but a further complication: As far as I
know, power meters are made to detect light that hits the sensor
orthogonally. So with a high NA lens, a lot of the incident light won't
be even detected.
I guess what one could do is to measure
a) the power without objective with a parked beam, focusing on a spot in
the center of the field of view. This would give the upper estimate but
not the true intensity since some of it is absorbed by the objective
itself. Transmittance is never 100%.
b) doing the same with the objective that is to be used. This will give
the lower estimate. Too low, since part of the light won't be measured
due to the incident angle.
The truth then is somewhere between the two values. With modern high NA
objectives which should have a high transmission my gut feeling is that
the truth would be closer to (a) than to (b).
You could take value (a) and correct it the transmission of the
objective at the given wavelength published by the manufacturer, if that
is available. But I don't think I have ever seen a paper that actually
did all that. Whatever value you take, as Andreas suggested you then
would have to relate it to the true pixel dwell time, i.e. disregarding
dead time of the scanner.
To get the exact value at the focal point in the sample also would
require to take the losses due to reflection at the coverslip into
account. In essence, I am definitely with Claire when she says:
> It is my
> thought that comparing relative powers on the same instrument is okay but
> comparing between systems will be very complex.
The Leica SP8 systems do allow to park the beam, as Craig suspected.
Since I always forget how to do that I put the procedure on our web
site, where I can easily find it :-)
http://www.bioimaging.bmc.med.uni-muenchen.de/manuals-protocols/maintenance_protocolls/laserpower/index.html
Cheers
Steffen
Am 19.07.2017 um 00:24 schrieb Craig Brideau:
> *****
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>
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>
> I've noted the effect Andreas mentions quite often. I usually set the
pixel
> dwell time to the maximum such that the laser will spend the longest time
> possible scanning out a single line, but even then the flyback can disturb
> the reading. The best way is to park the mirrors in the center position,
> although not all systems allow you to do that. Nikon's old C1 platform
> allows for it, and ThorLabs' ThorScanLS has a park button as well. I'm not
>> *****
>>
>> Hi Claire,
>> Confocals usually blank (switch off) the beam on the return and the power
>> meter averages between the on and off phases. Very slow scans are more
>> accurate an I usually use high zoom. Parking the beam is the better
option.
>>
>> Best wishes
>>
>> Andreas
>>
>> -----Original Message-----
>> From: "Claire Brown" <
[hidden email]>
>> Sent: 18/07/2017 18:21
>> To: "
[hidden email]" <
[hidden email]>
>> Subject: Re: Assessing phototoxicity in live fluorescence imaging
>>
>> *****
>> 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.
>> *****
>>
>> Thank you for this great article and pointing to many great resources.
>> I wanted to bring up one issue we have had when trying to work on
>> different microscope and compare light density/exposure.
>>
>> For the CLSM microscopes when we use a power meter at the focal plan the
>> power we measure depends a lot on the scan settings.
>> If we park the beam as a point we get one power. If we go to a 100x100
>> pixel array at zoom 1 with a 10x lens the power is different. if we
change
>> the scan speed the power is different again. I suspect this is related to
>> how the power meter integrates the light over time and also how sensitive
>> it is spatially across the sensor. We have decide to just quote our power
>> as the power we measure at the power meter with set conditions and we
>> detail those conditions in our materials and methods section of the
paper.
>> We try to use a 10x/0.3 planfluar lens with no phase optics if we can.
>>
>> We have stayed away from trying to calculate the power at the sample
>> because a lot of assumptions have to be made. The assumptions may be
>> different for wide-field versus CLSM versus light sheet versus spinning
>> disk and so on.
>>
>> We ran into these issues when just trying to repeat measurements on two
>> different confocals from two different manufacturers. It can really get
>> quite complex.
>>
>> Does anyone have thoughts on this issue? Any cleaver solutions? It is my
>> thought that comparing relative powers on the same instrument is okay but
>> comparing between systems will be very complex.
>>
>> Ideally, it would be good for the manufacturers to have some kind of
laser
>> power measurement in the instrument and software that is always
monitored.
>> Even if this is just a relative value to the actual power at the sample
it
>> would really improve quantitative microscopy and also help in maintenance
>> and trouble shooting equipment. I'm not sure about others but this kind
of
>> a feature would really be a strong selling point for me and the core
>> facilities I manage. In many cases these options are already built into
the
>> hardware for the service engineers but are not accessible to the end
user.
>>
>>
>> Sincerely,
>>
>> Claire
>>
--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging
Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany
http://www.bioimaging.bmc.med.uni-muenchen.de"