I am using cancer cell lines for my study. At lower magnifications of 20 and 40X, the stain is not affected even for long exposure time. However, at 60X, using oil, the stain bleaches in a matter of about 10 seconds. I am using fluoroshied to mount the slides.
Use confocal microscope for taking higher mag pictures because it has pin-hole exposure when compared to epifluorescent microscope (wide-field exposure).
Otherwise, use alexa fluor conjugated phalloidin (far better than classical dyes) if you desire to use epifluorescent scope.
Good luck
Thank you both for the suggestions.
Can anyone tell me how good is "ProLong® Gold Antifade Reagent"? n how does it compare to Fluorishield?
Since i don't have the option of getting alexa fluor conjugated phalloidin, i have to improve on the slide preparation and the necessary precautions..
Shrikant, Prolong works extremely well. My experiences is it acts better than Flourishield for protecting photobleaching of the 'red' dyes like Texas Red. It makes the signal appear brighter along with the background.
Imran, I agree that Texas Red and the longer wavelength Alexa dyes are much more photostable and will resist bleaching.
However, as for 'pinhole' exposure this is incorrect. The laser from point source scanner in the confocal will pass through the sample and photo bleach at least as effectively as wide-field epiflourescence with a mercury or xenon light source. The pinhole actually limits fluorescence detection by the photomultiplier tubes in the confocal. It's purpose is to limit detection of 'out of focus' light. To limit photobleaching, one must take single 'fast' scans while focusing and adjusting settings before starting high resolution image acquisition. This holds true for image acquisition by wide-field imaging with a cooled CCD camera.
Thank you Dr. Henley for detailed explanation. Experience always counts and every day one learns from peers. I hope this discussion will help others too when they come across such hurdles.
Regards
Imran
Dear Imran, As scientists we rely on the collective expertise of our community. I appreciate the time and effort that is shared by my colleagues to foster this experience.
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