I have a fluorescent light source and an objective lens and would like to measure the light intensity at the focal plane. I can measure the light intensity at a different position but need to calculate it at the focal plane. I am using a 40x, 1.3 NA, 0.2 mm WD lens. I guess I need to make an area calculation.
For context, I am using light to stimulate cells (on a coverslip) expressing an optogenetic construct and need to be able to estimate light intensity.
The first step is a light power meter, like a PM100D from Thorlabs with appropriate detector. Set that to the right wavelength, and read off the power in mW.
The you need to estimate the size of the excitation area. You probably have an etched calibration slide somewhere. Look down the scope, see what the diameter of your field of view is, and use 3.141*r^2 to know your area.
Divide the first measurement by the area, and your done.
Yes, the measurement of your excitation area will not be 100% accurate, but it will be close enough.
Thank you! I have the power measurement using a PMD100D(+ S130VC sensor) at a position ~ 2 mm from the focal plane and so I just manually measure the field of view through the eyepiece and calculate the mW/mm2 based on those two measurements. So simple!
Out of interest, is there a way of measuring the excitation area empirically (based on the objective specs)?
> is there a way of measuring the excitation area empirically?
Yes, it is *something* divided by the magnification of the objective. But what that *something* is ... well I'm not 100% certain.
The something is the whatever is the width limiting factor of your beam (assuming it is properly collimated). It's probably the back aperture of the objective. But if you're underfilling the back aperture, then it's something else. But still, the size of your back aperture, divided by the magnification is a pretty good place to start.
(1) Make efforts to learn more about radiometry and photometry basic terms and definitions.
(2) You can apply so called "camera equitation" to calculate irradiation in the lens focal plane starting from your object radiance (or intensity in the case of point source), geometrical relation between lens and your object and lens properties NA, magnification, input aperture, transmission.
(3) you can find related theory in any textbook related to electro-optics.
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