Hello,
When people are mentioning that for ex, Alexa fluorochromes are more photostable, I understand that they refering to their better photostability (reduced photobleaching) after a short (few sec to few min) illumination like during a microscopy or flow cytometry experiment. Am I correct?
what about repetitive illuminations of dyes? will they photodegrade or photobleach?
thanks for any comment.
I'm not quite sure what you're asking, but the more photo-stable the flurochrome means that you can image it multiple times with limited loss of brightness. The alexa dyes are very stable, they're most commonly used for immunofluorescence with powerful confocal lasers or super-resolution microscopes which take >300 images of a single area. They can also be stored for a long time without a significant loss in brightness so they can be imaged again in 12 months.
Dyes like FITC and PE are better used for applications like flow cytometry, where there is only a few exposures to a laser as these are less stable and bleach very quickly (reduction in brightness). If used for microscopy, if you have to take a z-stack (multiple images at different thickness) of tissue stained with PE, after the 10th or so exposure to the laser, there will be a significant reduction in the intensity of the dye, and you will need to adjust the exposure settings on the microscope.
Then there's brightness, which changes based on the flurochrome you're using. PE is very bright, 5 on a scale of 5. FITC is dim, about a 3/5 and Pacific Blue is even less, about a 1. Here is a chart from biolegend: http://www.biolegend.com/media_assets/support_resource/Flourochrome_Brightness_Index.pdf
If you want to do flow cytometry, Going to an introduction to flow course is a must. Immunofluorescence is fairly straightforward, this is a very standard protocol that most people would follow: http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol
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