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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