If the pinhole at the image plane is what rejects the out-of-focus fluorescence, then why does focused illumination matter? (I.e., why laser instead of widefield?) Is it to reduce interference from neighboring fluorescence?
You want to put your light in the place where it can be used. If you illuminated everything, very little light would return and you'd have to sit on each voxel for a long time.
Yes, all the other light wouldn't be useful (not collected through the aperture) and you'd like to maximize the signal from the point of interest. That part of your sample might only return 1 in a million photons (low reflectivity). @@allen8376
ok, some people at universties may label proteins but not me. You can also label proteins with radioactiv substances or normal dies specific. Fluorescence is not nessesary
