It is mentioned everywhere that fluorescent beads need to be sub resolution for empirically measure the point spread function (PSF). Why is this the case?
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The point spread function is the impulse response of the microscope. Therefore, to measure it, people make the image of a delta like object (an impulse in space), where the limit is set by the resolution and diffraction. If you use a bigger bead, its image will be composed by "many" psf, ie by the convolution of the real object with the psf.
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$\begingroup$ You don't need a delta function for image deconvolution to determine the PSF. That's just one way of many. $\endgroup$ Commented Jan 10, 2016 at 16:25
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$\begingroup$ @CuriousOne reworded. The question is not about methods to measure the PSF, but it can be interesting if you could list the other ways you mention. $\endgroup$– scrx2Commented Jan 10, 2016 at 17:07
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$\begingroup$ @fxpd: The OP posted a similar question and I gave a reference there. A simple google search brings up quite a number of papers. $\endgroup$ Commented Jan 10, 2016 at 17:10