Re: Quenching of 2p excited DAPI with GFP?

Posted by Page Baluch on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quenching-of-2p-excited-DAPI-with-GFP-tp592996p592997.html

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Merek,
Maybe you could try using a different DNA stain like Draq5? It excites
with 633 (He/Ne) and emits in the far red so the signal will be well
separated from the gfp and might avoid any FRET like energy transfer.

Page

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Merek Siu
Sent: Tuesday, June 10, 2008 11:18 AM
To: [hidden email]
Subject: Quenching of 2p excited DAPI with GFP?

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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