# PSF Measurements In Fluorescence Imaging

Quite a technical question!

I have measured the Point Spread Function of 100nm fluorescent breads with my Olympus scanning head. I'm two-photon exciting the beads with a wavelength of 800nm and focused in the sample with a 100x with N.A.: 1.4

The theory suggests me the resolution of such a system to be: $$d=\frac{0.7\cdot\lambda}{N.A.}\approx 400nm$$

Now that value should be equal (with at most a 18% correction) to the FWHM of the 2D gaussian I obtain on the image.

But from my analysis of the images I obtain a FWHM close to 2um. Now surely the formula is for an ideal optical setup but a factor five seems to me quite strange! Is that possible to obtain such a result, in the case of very not ideal optical elements, or should I look for some sort of problem with the acquisition sw that tells me the pixel dimension of the images?

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