Hi everyone,
My problem is quite simple: I would like to get the fluorescence excitation intensity in mol.m-2.s-1 (or W.m-2). And I didn't find any protocol/advice online to measure it.
I typically use GFP or mCherry as fluorescent protein.
What I have done is measure the overall power (in W) of the microscope beam using a PM100D power meter (Thorlabs) and a S120C power sensor, while varing the input intensity (U.A) in the software. The response is not perfectly linear but that's fine I can compensate for that.
What I would like is to get the power flux density or intensity (I'm not sure about these names). To do so I just need the area of illumination and use this formula: intensity=power/area.
The problem is that I don't know how to measure/calculate the beam area of illumination, do you have any advice?
The way I want to measure this excitation intensity may be totally wrong, and I'm very surprised that I don't find any clue on how to do it, while a lot of people use this unit.
Thanks for anyone who could help me with this issue.
Matthias LE BEC
You need to close down the microscope field stop so that you are illuminating a defined area, like your camera chip, with the lens you will be using for imaging. Close the stop down so that it is just outside the field of view. If you know the physical pixel size of your camera, you can calculate the area being illuminated. So my camera region I image with is 256x256 pixels. The physical pixel size is 16 um, with a 100X lens this gives me me 16um/100 = 0.16 um/pixel. So my area is (256(0.16))2 or 1677 um2
Hold your power meter close to the lens because the light is highly divergent
Thank you for your reactivity and precision !
This seems perfect, I will try it as soon as possible, and let you know if this work for me.
Thanks again :)
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