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Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation
P C Peters1, C Thoni, E J Harry
1Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, 2007, Australia. phoebe.c.peters@student.uts.edu.au
Abstract:
A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.
