# 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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## 2 Answers

You have many things which can go wrong when you try to go down to the wavelength limit, without resorting to Very not ideal optical element : non-Gaussian beam, wrong wavelength, spherical aberration, etc. I fear there is no easy answer to your question.

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no well, if someone else tells me he obtained a result similar (factor 5) i will be satisfied.. cause i've never really take a measurements like this and i don't know if it is normal or i have some technical problem in the analysis.. thx – Steve Nov 17 '10 at 10:20

ok i've found the solution to my problem.. first of all i had a factor 2 discrepancy due to the pixel dimension and, more important from an optical point of view, the laser beam was underfilling the entrance pupil of the objective: i.e. the focusing was worse!

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