Nuclear stain that is not excited by 1050nm in two photon

[Shalin Mehta]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi everyone,
Is there a nuclear stain that is NOT excited by 1050nm wavelength in
two photon, but excitable by 850-900nm. I have been imaging Hoechst,
AF488, RFP labeled embryos.  1050nm  is needed to excite AF488 and
RFP. This is far away from Hoechst's optimal excitation wavelength of
820nm, but Hoechst label is pretty bright. Hoeschst's emission
spectrum is too broad and swamps the AF488 channel.

Alternatively, a stain which has narrow emission spectrum in blue will
be helpful.

Thanks for the ideas
Shalin

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.

[Michael Giacomelli]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I've tried DAPI at 1 um and seen virtually no signal (optimal is about 750
nm), although its not too different from Hoechst.  Worth a try at least.

Mike

On Fri, Oct 17, 2014 at 10:32 AM, Shalin Mehta <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi everyone,
> Is there a nuclear stain that is NOT excited by 1050nm wavelength in
> two photon, but excitable by 850-900nm. I have been imaging Hoechst,
> AF488, RFP labeled embryos.  1050nm  is needed to excite AF488 and
> RFP. This is far away from Hoechst's optimal excitation wavelength of
> 820nm, but Hoechst label is pretty bright. Hoeschst's emission
> spectrum is too broad and swamps the AF488 channel.
>
> Alternatively, a stain which has narrow emission spectrum in blue will
> be helpful.
>
> Thanks for the ideas
> Shalin
>
> Assistant Research Scientist,
> Marine Biological Laboratory,
> 7 MBL Street, Woods Hole MA 02543, USA
>
> website: http://mshalin.com
> (office) Lillie 110, (ph) 508-289-7374.
>

[Guy Cox-2]

Shalin,

        I think you are probably seeing 3-photon excitation of your Hoechst.  Can you use the stain at much lower concentration?  Otherwise you might try one of the far-red DNA dyes such as TOTO-3 or TOPRO-3.  Their emission peak is about 50nm longer that RFP so youj should be able to separate them on the emission side.

                                                              Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Shalin Mehta
Sent: Saturday, 18 October 2014 1:32 AM
To: [hidden email]
Subject: Nuclear stain that is not excited by 1050nm in two photon

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi everyone,
Is there a nuclear stain that is NOT excited by 1050nm wavelength in two photon, but excitable by 850-900nm. I have been imaging Hoechst, AF488, RFP labeled embryos.  1050nm  is needed to excite AF488 and RFP. This is far away from Hoechst's optimal excitation wavelength of 820nm, but Hoechst label is pretty bright. Hoeschst's emission spectrum is too broad and swamps the AF488 channel.

Alternatively, a stain which has narrow emission spectrum in blue will be helpful.

Thanks for the ideas
Shalin

Assistant Research Scientist,
Marine Biological Laboratory,
7 MBL Street, Woods Hole MA 02543, USA

website: http://mshalin.com
(office) Lillie 110, (ph) 508-289-7374.