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Hoechst 32258 two photon excitation spectra

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david anchel

Hoechst 32258 two photon excitation spectra

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

I was wondering if anyone could tell me the TWO PHOTON excitation spectra for Hoechst 32258? I would like to excite a second fluorophore with my two photon tuned to between 700nm and 930nm without also exciting the Hoechst 32258. I would like to be sure that I am exciting at a wavelength that is well outside of the two photon Hoechst 32258 excitation spectra, and between 700nm and 930nm. Does anyone also know the two photon excitation spectra of Alexa 430? I am considering using this as the second fluorophore but am unsure if it can be excited outside the excitation spectra of Hoechst 32258 with the two photon tuned to 930nm or less.

Thanks,
Dave anchel,
Toronto

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Mayandi Sivaguru

Re: Hoechst 32258 two photon excitation spectra

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dave, I think you mean Hoechst 33258, I don't think you can excite this dye and the Alexa 430 with two different 2P absoption lambda between 700-930 nm. Since the absorption and emission spectra (single photon) of this dye and the DAPI is pretty similar (you might even get a FRET effect since the emission lambda of Hoechst is overlapping nicely with Alexa 430), you can excite this Hoechst at around 760-800 nm. But if the sample also contains Alexa 430, provided the target for this Alexa is not localized in the nucleus (elsewhere in the cell), then you can get an image with nucleus (Hoechst) and elsewhere labeled (Alexa 430). If this is not the case (in case your target localizes exactly inside the nucleus or DNA), then you have to try this in a spectral detector or switch the probes, if possible.
Shiv

At 01:27 PM 2/24/2008, you wrote:

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

I was wondering if anyone could tell me the TWO PHOTON excitation spectra for Hoechst 32258? I would like to excite a second fluorophore with my two photon tuned to between 700nm and 930nm without also exciting the Hoechst 32258. I would like to be sure that I am exciting at a wavelength that is well outside of the two photon Hoechst 32258 excitation spectra, and between 700nm and 930nm. Does anyone also know the two photon excitation spectra of Alexa 430? I am considering using this as the second fluorophore but am unsure if it can be excited outside the excitation spectra of Hoechst 32258 with the two photon tuned to 930nm or less.

Thanks,
Dave anchel,
Toronto

<html><DIV>
<DIV></DIV></DIV></html>

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: 217.244.2496
[hidden email]
http://core.igb.uiuc.edu

Armstrong, Brian

Re: Hoechst 32258 two photon excitation spectra

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

Hi Dave, I often excite Hoescht at 730nm and my red fluorophore (it varies) at 860nm – 900nm. I am not getting excitation of the Hoescht at 860 -900 and I am not exciting my reds at 730. Cheers,

Brian Armstrong PhD

Manager, Light Microscopy Core

Beckman Research Institute

1450 East Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Tuesday, February 26, 2008 11:02 AM
To: [hidden email]
Subject: Re: Hoechst 32258 two photon excitation spectra

Dave, I think you mean Hoechst 33258, I don't think you can excite this dye and the Alexa 430 with two different 2P absoption lambda between 700-930 nm. Since the absorption and emission spectra (single photon) of this dye and the DAPI is pretty similar (you might even get a FRET effect since the emission lambda of Hoechst is overlapping nicely with Alexa 430), you can excite this Hoechst at around 760-800 nm. But if the sample also contains Alexa 430, provided the target for this Alexa is not localized in the nucleus (elsewhere in the cell), then you can get an image with nucleus (Hoechst) and elsewhere labeled (Alexa 430). If this is not the case (in case your target localizes exactly inside the nucleus or DNA), then you have to try this in a spectral detector or switch the probes, if possible.
Shiv

At 01:27 PM 2/24/2008, you wrote:

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Haberman, Ann

Re: Hoechst 32258 two photon excitation spectra

In reply to this post by david anchel

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Re: Hoechst 32258 two photon excitation spectra

Hi Dave,

Although I have not tried other Hoechst variants, I have found that Hoechst 33342 can be excited at fairly long wavelengths. I have routinely imaged this Hoeschst at wavelengths as long as 865 nm, which has allowed me to simultaneously excite GFP.

-Ann Haberman

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

I was wondering if anyone could tell me the TWO PHOTON excitation spectra for Hoechst 32258? I would like to excite a second fluorophore with my two photon tuned to between 700nm and 930nm without also exciting the Hoechst 32258. I would like to be sure that I am exciting at a wavelength that is well outside of the two photon Hoechst 32258 excitation spectra, and between 700nm and 930nm. Does anyone also know the two photon excitation spectra of Alexa 430? I am considering using this as the second fluorophore but am unsure if it can be excited outside the excitation spectra of Hoechst 32258 with the two photon tuned to 930nm or less.

Thanks,
Dave anchel,
Toronto

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--

Ann Haberman, PhD
Connecticut Microscopy Society, President
In Vivo Imaging Facility, Director

Department of Laboratory Medicine
Yale University School of Medicine
300 Cedar Street
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

david anchel

Re: Hoechst 32258 two photon excitation spectra

In reply to this post by Armstrong, Brian

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

What reds (I am hoping to use an Alexa to minimize photbleaching) do you use in particular to excite at 860-900 that do not excite the Hoechst?

Dave

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Date: Tue, 26 Feb 2008 11:38:58 -0800
From: [hidden email]
Subject: Re: Hoechst 32258 two photon excitation spectra
To: [hidden email]

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

Hi Dave, I often excite Hoescht at 730nm and my red fluorophore (it varies) at 860nm – 900nm. I am not getting excitation of the Hoescht at 860 -900 and I am not exciting my reds at 730. Cheers,

Brian Armstrong PhD

Manager, Light Microscopy Core

Beckman Research Institute

1450 East Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Tuesday, February 26, 2008 11:02 AM
To: [hidden email]
Subject: Re: Hoechst 32258 two photon excitation spectra

Dave, I think you mean Hoechst 33258, I don't think you can excite this dye and the Alexa 430 with two different 2P absoption lambda between 700-930 nm. Since the absorption and emission spectra (single photon) of this dye and the DAPI is pretty similar (you might even get a FRET effect since the emission lambda of Hoechst is overlapping nicely with Alexa 430), you can excite this Hoechst at around 760-800 nm. But if the sample also contains Alexa 430, provided the target for this Alexa is not localized in the nucleus (elsewhere in the cell), then you can get an image with nucleus (Hoechst) and elsewhere labeled (Alexa 430). If this is not the case (in case your target localizes exactly inside the nucleus or DNA), then you have to try this in a spectral detector or switch the probes, if possible.
Shiv

At 01:27 PM 2/24/2008, you wrote:

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

I was wondering if anyone could tell me the TWO PHOTON excitation spectra for Hoechst 32258? I would like to excite a second fluorophore with my two photon tuned to between 700nm and 930nm without also exciting the Hoechst 32258. I would like to be sure that I am exciting at a wavelength that is well outside of the two photon Hoechst 32258 excitation spectra, and between 700nm and 930nm. Does anyone also know the two photon excitation spectra of Alexa 430? I am considering using this as the second fluorophore but am unsure if it can be excited outside the excitation spectra of Hoechst 32258 with the two photon tuned to 930nm or less.

Thanks,
Dave anchel,
Toronto

<html><DIV>
<DIV></DIV></DIV></html>

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: 217.244.2496
[hidden email]
http://core.igb.uiuc.edu

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Armstrong, Brian

Re: Hoechst 32258 two photon excitation spectra

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

Hi, I have mainly been using dextran rhodamine in intravital imaging. However, it is the wavelength you are most interested in. I have not seen Hoescht excitation at 860nm, but I believe Ann said she did. So, you need to see for yourself on your own sample, it may also depend on depth and percent laser power. You should get nice excitation from the red Alexas although I have not tried them yet. My guess is that you should be able to find a wavelength through empirical testing with nice excitation of your red without exciting your Hoescht.

Cheers,

Brian Armstrong PhD

Manager, Light Microscopy Core

Beckman Research Institute

1450 East Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of david anchel
Sent: Tuesday, February 26, 2008 2:30 PM
To: [hidden email]
Subject: Re: Hoechst 32258 two photon excitation spectra

Hi there Brian,

What reds (I am hoping to use an Alexa to minimize photbleaching) do you use in particular to excite at 860-900 that do not excite the Hoechst?

Dave

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Date: Tue, 26 Feb 2008 11:38:58 -0800
From: [hidden email]
Subject: Re: Hoechst 32258 two photon excitation spectra
To: [hidden email]

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

Hi Dave, I often excite Hoescht at 730nm and my red fluorophore (it varies) at 860nm – 900nm. I am not getting excitation of the Hoescht at 860 -900 and I am not exciting my reds at 730. Cheers,

Brian Armstrong PhD

Manager, Light Microscopy Core

Beckman Research Institute

1450 East Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/SharedResources/LightMicroscopy/LightMicroHome.htm

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