Muhammad Anzar wrote- >We are working on the uptake of a rhodamine-labeled protein by >spermatozoa. Under fluorescent microscope, there is clear fluorescence >on sperm head but but it can not be detected by flow cytometer (argon >ion laser of 488 nm) using FL3 (wavelength 580). Two different flow >cytometers have been tried but the results are same. Your suggestions in >solving this problem would be highly appreciated. Rhodamine has almost no absorption at 488 nm; when you look at it under a fluorescence microscope, you normally use green excitation (e.g., the 546 nm line from a mercury lamp). If you have a lot of rhodamine on a cell, you may be able to see it with 488 nm excitation; otherwise, you'd need a green excitation source, and, while argon laser emission at 515 nm would give some excitation of rhodamine, the argon lasers in most benchtop flow cytometers cannot be adjusted to emit this wavelength. Your best bet would be to use a different label; otherwise, you will need to use a flow cytometer with green excitation. A large (sorting) instrument with a big air-cooled or a water-cooled argon laser would allow you to use the 515 nm line; you would do better with 520 nm or 530 nm from a krypton laser. 532 nm from a doubled YAG laser, or 543 nm from a green He-Ne laser would also work; however, the green He-Ne's only put out about 2 mW and thus are practical only in cytometers with very efficient light collection optics. -Howard
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