Posted by
Glyn Nelson on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DNA-dyes-tp592122p592131.html
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalMy experience agrees with Gert. I've successfully imaged cells with
hoechst using 780 nm for 2 photon and still seen cell division. This was
in MEFs and HeLa cells. I was also imaging GFP, RFP and a far red dye in
the same cells, and could keep them alive with division for 3 days
(probably longer, but I didn't try). Obviously the key is to minimise
light input. I titred down the amount of hoechst added so that I kept
cells alive (same division rate as controls), and added the minimum of
light to the system. At the dose I used, I saw no effect on cell viability
either with or without 780nm excitation. In my experience, there is
absolutely no way of performing live cell imaging with UV excitation; if
you want blue dyes you need to use 2 photon. UV causes too much cellular
damage, even if you keep the input low enough so that the cells survive,
you are causing too much cellular damage to trust your data.
So I would titre the hoechst to a satisfactory level for your cell line,
and use a 2 photon. If you don't have access to one, histone-FPs are a
good alternative and move you away from UV excitation (assuming your cells
are transfectable).