Regarding pixel size, if the pixel size is too large (> 500nm) then most or all of the light emitted by a single fluorescent dye molecule will be captured by a single pixel. In this case your resolution will be limited by your pixel size, and will be very low, technically 2x the pixel size due to the Nyquist sampling limit. If the pixel is too small (< 20nm) the fluorescence from the dye molecule will be spread across many pixels, so the ratio of signal to read noise will be correspondingly lower. The value of 100nm is a compromise between these two regimes. The exact value will depend on the specifics of the camera, and can be calculated. I believe there are papers on this subject, perhaps from the Ober lab (https://www.wardoberlab.com/)? However, the minima here is relatively broad, so getting the pixel size exactly right isn’t super important as long as it is close. I’ve used setups that had pixel sizes between 160nm and 80nm without noticing much difference in resolution.