Re: Quenching of 2p excited DAPI with GFP? (manufacturer input)

[Roy Edward]

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Dear Merek

To add to Debra's suggestion there is some recent data on 2-P with DRAQ5:

Smith PJ, et al. Cytometry 40:280–291 (2000) (see fig. 5)

Best regards

Roy Edward

 

Biostatus Ltd

TEL: [phone]; [hidden email]; www.biostatus.com

Co. #: 3079239. Regd. in England & Wales. Regd. office: 56 Charnwood Road, Shepshed, LE12 9NP, UK.

---

From: Debra Baluch [mailto:[hidden email]]
Sent: Tue 10/06/2008 23:38
Subject: Re: Quenching of 2p excited DAPI with GFP?

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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 [[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

[Mayandi Sivaguru]

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Merek, take out the vectashield and use the DAPI in aqueous solution at 10-50 microgram/mL in PBS for 30 min and wash for 3x 2 min each in PBS and mount in an appropriate antifade mounting media and try with the same set up, my understanding is that you are using 800 nm excitation on your 2P for DAPI. You are also right regarding the absorption of GFP in the same lambda as DAPI, but the one I suggest is despite vectashield DAPI works for some samples, it doesn't in a bunch of other samples due to DAPI readily and highly penetrate cells when it is in aqueous solution rather than a denser media with glycerol. Contact me off the list should you have any questions.
Thanks
Shiv

At 03:55 AM 6/11/2008, you wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Merek
To add to Debra's suggestion there is some recent data on 2-P with DRAQ5:
Smith PJ, et al. Cytometry 40:280–291 (2000) (see fig. 5)
Best regards
Roy Edward<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />
 
Biostatus Ltd
TEL: [phone]; [hidden email]; www.biostatus.com
Co. #: 3079239. Regd. in England & Wales. Regd. office: 56 Charnwood Road, Shepshed, LE12 9NP, UK.

---

From: Debra Baluch [[hidden email]]
Sent: Tue 10/06/2008 23:38
Subject: Re: Quenching of 2p excited DAPI with GFP?

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

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 [[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

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
[hidden email]
http://core.igb.uiuc.edu