Using reference objects to estimate the point spread function?

1. How do you measure PSF on a microscope?
2. How do I get PSF from an image?
3. What affects point spread function?

How do you measure PSF on a microscope?

The PSF is often measured using a fluorescent bead embedded in a gel that approximates an infinitely small point object in a homogeneous medium.

How do I get PSF from an image?

To be sure, you can calculate it knowing the optical resolution d = lambda / 2*NA. (NA is the second number written on the objective lens, after the slash after the magnification number.) Then divide d by the pixel spacing in the image and that is the approximate PSF size.

What affects point spread function?

So What Affects the Point Spread Function? The PSF varies depending on the wavelength of the light you are viewing: shorter wavelengths of light (such as blue light, 450 nm) result in a smaller PSF, while longer wavelengths (such as red light, 650 nm) result in a larger PSF and, therefore, worse resolution.