Hello, you could coat yourself the beads with an appropriate dye. Regarding the bead size, it depends also on the magnification of your objective. 200nm should be ok for a 63x or 100x objective. if you use a lower magnification, you might want to use larger beads.

Hello Ahmed,

I assume you are using a confocal microscope, so you should be able to use any emission wavelength you want, as the resolution should mostly depend on the excitation beam. If that is the case, check the specific excitation spectra of the green-yellow beads you find. While the maximum of absorption will probably be at 488, beads are generally very bright, and have quite wide excitation spectra, so you should be definitely fine using them with a 440 laser, and probably even at 405.

As for the bead size, you should be fine as long as it is smaller than the diffraction limit (wavelength divided two times the NA). The very important part is for them to be very bright, not necessarily big, so in case try to slower your scan speed as much as possible for the measurement.

Also, let me introduce to a very important secret in microscopy: While for published data you should always use the proper calibrated beads from a reliable source, for internal testing you can try to dissolve the ink of Stabilo highlighter pen in water (it has to be Stabilo branded, it never worked for me with pther brands). The ink is slightly hydrophobic, and will form very bright sub-diffraction aggregates. That's a good source of very cheap "microbeads" in different colors.

Hello,

As an alternative approach to the above suggestions is that you can also measure this using a thin fluorescent layer, the advantage is that it gives access to the psf (in z-direction) in the whole field of view. I am out of the field now, I don't know if they are currently commercially available, but this is described in:

Article Characterization of sectioning fluorescence microscopy with ...