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I am using a microscope with an LED derived light through the epi-fluorescent port of a microscope. I know that the "field of view" for a given objective is equal to the field number/magnification but I don't know that this is the actual area of illumination on the sample. In my case, I am using a 40x objective with field number 26.5.

Secondly, if I were to use a pinhole filter between the light source and the back of the objective would this limit the area of illumination so long as the pinhole was smaller than the f.o.v. dimension?

I would be grateful for any answers, most of the information I can find on beam size at sample is related to spot size calculations of resolution.

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Well you can always use a piece of white circular paper and draw the outline of the light onto it since it should be visible. This is a quick and dirty method that can be improved by taking a photo of the light with a camera if you have one and then comparing the distance of the edge of the paper measured with a ruler to that of the circle of light.

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