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
Thank you so much for this explanation! It is much much clearer now. I am so happy that I found this video.
Thanks a lot for the video. The explanation is crystal clear and easy to follow! Thanks for sharing.
Clear and outstanding elaboration.
Thank you, best explanation
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.
@@roadrunner6653 so it’s for the intensity of light at the point of interest?
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
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much blah-blah and no results. DIC is the better solution because fluorescence-microscopy still gives unsharp images
Dude your words make no sense. How do you label a specific protein in DIC?
@@testboga5991 why should i label proteins, only Dudes label proteins
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