# How do I calculate the illuminated spot on specimen in optical microscope?

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1. Nov 27, 2012

### tr111

Hi,

When we use an optical microscope, how do we roughly calculate the illuminated spot size on the sample ? Also what is the light power on that spot?

And what if I change to fluorescence microscope(epi illumination)? Cause the illuminating light go through the rear side of objective to the surface of specimen, also how do we calculate the spot size?

Thanks!

2. Nov 27, 2012

### Simon Bridge

Welcome to PF;
In both cases, we use our knowledge of the optics of the microscope in question.
Different microscopes will have different optical properties so I cannot be exact.

Of course - usually you control the illumination independently of the microscope - though the microscope will only have a limited area in it's field of view. It would help a lot if you told us the situation you are thinking of.

3. Nov 28, 2012

### tr111

I would like to build a fluorescence measurement setup as simple as possible,just like this one,

got the filters needed, and an objective lens, even one more convex lens for tube lens as in microscope but without any diaphragms, not that complicated as commercial fluorescence microscope. what I would like to build, for example, the light source is already collimated parallel lights with diameter = 7mm. the objective lens is 10X, parfocal length=45mm, NA=0.25, WD=10mm. when the excitation light reflects by dicroic mirror and hits the rear side of objective, because of the pupil diameter of objective=2*NA*f ~5.3mm here(f is the focal length of objective, I assume the value is close to WD), only part of incoming excitation light can go through the objective lens to illuminate the specimen(assume it is 1cm*1cm). and then I don't know how to calculate how much part of specimen will be shined.... And I do not know how to roughly calculate the light intensity(watt) of this illuminated spot either.

I had done google it so hard, but I cannot find anyone doing this kind of quantitative work.
I am not familiar with optics, so I am not sure this calculating idea whether is doable or not..

Thanks.

4. Nov 28, 2012

### tr111

cause what I like to build is so simple, I have to do this calculation first to make sure later on the intensity of fluorescence of my sample that hit on the camera could be saw by the CCD sensor.

5. Nov 28, 2012

### Simon Bridge

With all the collimated light passing through the lens, then all of it arrives in the illuminated spot. So you need the intensity of the light in watts per square meter that enters the objective ... this, multiplied by the area of the spot is the incedent light power.

It is a converging lens - so parallel light converges to the focus (except close to the focus where it forms a waist) ... so the size of the spot depends on the distance to the surface. It is simple geometry from the diameter of the (bottom) lens and the focal length.

The light from there will be scattered depending on the surface ... if you assume this is even then the proportion that enters back into the objective is just the area of the bottom lens divided by the area of the hemisphere at that distance.

Then you are just running the optics backwards.

Cool project :)