Quenching of 2p excited DAPI with GFP?

Posted by Merek Siu on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quenching-of-2p-excited-DAPI-with-GFP-tp592996.html

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Dear all,

We have some users who are looking at GFP-transfected neurons (fixed in
paraformaldehyde) that are mounted with Vecta-Shield containing DAPI. Since
we do not have a UV laser on our Leica SP5, we sequentially scan for GFP
first (one photon excitation at 488 nm), and secondly followed by a DAPI
image using two photon excitation at 760 nm.

Although the DAPI signal is strong in non-transfected cells, in
GFP-transfected cells (where the GFP should be cytosolic) the DAPI signal is
much weaker or similar to background. Under epifluorescence with a mercury
lamp, both the DAPI and GFP signals are strong for the transfected cells.

My guess is that the blue emission from the nuclear DAPI is being mostly
absorbed by the cytosolic GFP. Does that sound right?

Can anyone suggest an imaging approach to get around the problem (e.g.
different excitation. Other approaches could be different FPs or DNA dyes)?

Out of curiosity, do people see similar behavior with one photon excitation
of DAPI using a UV laser?

Thanks!
Merek Siu