Quenching of 2p excited DAPI with GFP?

[Merek Siu]

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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

[Rosemary.White]

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Unless you have bad autofluorescence in the red, you could use propidium
iodide which stains DNA and fluoresces in the red with 488 nm excitation.
cheers,
Rosemary

Rosemary White                    [hidden email]
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Australia


On 11/6/08 4:18 AM, "Merek Siu" <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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

[Mario-2]

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Merek,

Maybe I am just tired (end of term), but the variation of the "inner
filter effect" you allude to doesn't seem to square with the standard
epi- experiment where you can see both DAPI and GFP signals.

1. Are you certain that you are using the proper emission filter with
the multiphoton excitation? DAPI has quite a broad emission
(bluish-white looking). The more the filter selects for blue-violet
emission the less interference from GFP.

2. In terms of excitation, have you checked that 760 nm excitation
does not excite the GFP you are using? A very rough estimate for
excitation for GFP using 760 nm will still be several percent of the
peak. You did not mention the concentration of DAPI you used but you
might try increasing it. Some folks use at little as 0.1 ug/ml but
FACS applications use up to 3 ug/ml. Maybe something in between would
be worth trying (I like 0.2-0.5 ug/ml).

If absorption of the emitted DAPI fluorescence is significant, you
would need the equivalent of 10, 100, or higher mM GFP in cytosol.
That seems extreme to say the least.

I would be interested to know what you come up with,

Mario

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]
[hidden email]

[Page Baluch]

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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?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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