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Hi Richard,
I've seen this with some far red dyes, such as mitotrackers. It's most
likely due to single-photon absorption of your MP laser wavelength.
When you look at single-photon absorption curve of DY-650, it's nearly zero
at 700-710 nm, but not exactly zero. Together with the fact that the MP
laser light is typically three orders of magnitude stronger than a
corresponding 1P laser (e.g. 633 nm), it can cause rapid photobleaching.
Obvious solution is to take the DY-650 image (or stack) first and then the
MP image. You should also try longer MP wavelength (e.g. 760 nm), that will
still excite Hoechst, but may not bleach the DY-650 so quickly...
Good luck!
Best, zdenek
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth
email:
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---------- Původní e-mail ----------
Od: Cole, Richard W (HEALTH) <
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Komu:
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Datum: 12. 2. 2018 12:08:31
Předmět: DY650 & multiphoton
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Dear Imagers,
I am trying to image DY650 (1P) and Hoechst with MP (710nm), surprisingly
the MP seems to be bleaching the DY650 even in sequential scans. Anyone else
experiencing this? I don't see any 2P spectra for the DY series?
Thanks
Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York
120 New Scotland Avenue, Albany N.Y. 12208
518-474-7048 Phone
518-408-1730 Fax
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