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mCherry and 2P

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Koo, Lily (NIH/NIAID) [E]

mCherry and 2P

Dear Listers,

I am wondering if any of you have had success imaging mCherry in the 2-photon mode? We have mCherry bacteria rods that are very bright in confocal, but cannot get an image in 2P. We tried up to 1030nm (the coherent laser stops at 1040 and the power drops down to ~600mW at 1030), but with no success. Around 900nm we could detect a shadowy image of the bacteria, but at a power level that immediately bleaches the fluorophore.

Any suggestions?

Thanks,

Lily

Quoc Thang Nguyen

Re: mCherry and 2P

> Dear Listers,
>
> I am wondering if any of you have had success imaging mCherry in the
> 2-photon mode? We have mCherry bacteria rods that are very bright in
> confocal, but cannot get an image in 2P. We tried up to 1030nm (the
> coherent laser stops at 1040 and the power drops down to ~600mW at 1030),
> but with no success. Around 900nm we could detect a shadowy image of the
> bacteria, but at a power level that immediately bleaches the fluorophore.
>
> Any suggestions?
>
> Thanks,
>
> Lily
>

Try 740-760 nm. Works great.

Greg Martin-8

Re: mCherry and 2P

In reply to this post by Koo, Lily (NIH/NIAID) [E]

Hey Lily --

I just did some 2-photon imaging of mCherry at 1000nm and it worked great with little bleaching. This was on fixed mammalian cells with the Spect. Phys. HP deep see laser with 15% transmission. Sorry, don't have a real number for power for comparison. Do you have non-descanned detectors on your system? Have you been able to image any other red fluors with your 2-photon?

Be peace! Greg.

Greg Martin

Keck Microscopy Facility
University of Washington
www.depts.washington.edu/keck

Lynn and Mike Garvey Cell Imaging Laboratory
University of Washington Institute for Stem Cell and Regenerative Medicine

206-685-8784 (office)
425-344-2632 (cell)

----- Original Message -----

From: "Koo, Lily (NIH/NIAID) [E]" <[hidden email]>

To: <[hidden email]>

Sent: Tuesday, June 23, 2009 11:26 AM

Subject: mCherry and 2P

Rainer Kohler-2

Re: mCherry and 2P

In reply to this post by Koo, Lily (NIH/NIAID) [E]

Hi Lily,

I recently imaged an mCherry fusion protein in Hela cells with a Mai
Tai DeepSee laser on an Olympus Fv1000 system. It had a maximum
signal at around 760 nm.

Best regards

Rainer

>Dear Listers,
>
>I am wondering if any of you have had success imaging mCherry in the
>2-photon mode? We have mCherry bacteria rods that are very bright
>in confocal, but cannot get an image in 2P. We tried up to 1030nm
>(the coherent laser stops at 1040 and the power drops down to ~600mW
>at 1030), but with no success. Around 900nm we could detect a
>shadowy image of the bacteria, but at a power level that immediately
>bleaches the fluorophore.
>
>Any suggestions?
>
>Thanks,
>
>Lily

--
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Guy Cox

Re: mCherry and 2P

In reply to this post by Koo, Lily (NIH/NIAID) [E]

I've never tried this but looking at all the other replies my
guess would be that your IR blocking filter in the MP is blocking
the mCherry fluorescence. Check out what that filter is. I certainly
find that our blocking filter on our Leica SP2 MP blocks chlorophyll
fluorescence, for example. What is your blocking filter?

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Wednesday, 24 June 2009 4:26 AM
To: [hidden email]
Subject: mCherry and 2P

Dear Listers,

I am wondering if any of you have had success imaging mCherry in the 2-photon mode? We have mCherry bacteria rods that are very bright in confocal, but cannot get an image in 2P. We tried up to 1030nm (the coherent laser stops at 1040 and the power drops down to ~600mW at 1030), but with no success. Around 900nm we could detect a shadowy image of the bacteria, but at a power level that immediately bleaches the fluorophore.

Any suggestions?

Thanks,

Lily

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM

Koo, Lily (NIH/NIAID) [E]

Re: mCherry and 2P

Thanks for all the replies. I am working on our collaborator's Meta510 and just getting warmed up to the system. They do have a non-descanned detector, which didn't help us in capturing mCherry signal when we tried from 800-1030. At 800, we started to see some signal, but there was significant bleaching. I believe the blocking filter is set at 690nm. We will try 740nm ex. tomorrow and will let you all know how it will work out.

Thanks,

Lily

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox [[hidden email]]
Sent: Tuesday, June 23, 2009 9:41 PM
To: [hidden email]
Subject: Re: mCherry and 2P

I've never tried this but looking at all the other replies my
guess would be that your IR blocking filter in the MP is blocking
the mCherry fluorescence. Check out what that filter is. I certainly
find that our blocking filter on our Leica SP2 MP blocks chlorophyll
fluorescence, for example. What is your blocking filter?

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Wednesday, 24 June 2009 4:26 AM
To: [hidden email]
Subject: mCherry and 2P

Dear Listers,

I am wondering if any of you have had success imaging mCherry in the 2-photon mode? We have mCherry bacteria rods that are very bright in confocal, but cannot get an image in 2P. We tried up to 1030nm (the coherent laser stops at 1040 and the power drops down to ~600mW at 1030), but with no success. Around 900nm we could detect a shadowy image of the bacteria, but at a power level that immediately bleaches the fluorophore.

Any suggestions?

Thanks,

Lily

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM

Koo, Lily (NIH/NIAID) [E]

Re: mCherry and 2P

Dear all,

I would like to inform the list that we had good signal on mCherry when we excited it around 740nm. The signal dropped significantly after 790 (we hope to excite both mCherry and GFP). Our prior lack of success was apparently due to the fact that we were trying it on fixed samples (mCherry labelled bacteria), which killed the emission nearly entirely.

Thanks,

Lily

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Wednesday, June 24, 2009 1:23 AM
To: [hidden email]
Subject: Re: mCherry and 2P

Thanks for all the replies. I am working on our collaborator's Meta510 and just getting warmed up to the system. They do have a non-descanned detector, which didn't help us in capturing mCherry signal when we tried from 800-1030. At 800, we started to see some signal, but there was significant bleaching. I believe the blocking filter is set at 690nm. We will try 740nm ex. tomorrow and will let you all know how it will work out.

Thanks,

Lily

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Guy Cox [[hidden email]]
Sent: Tuesday, June 23, 2009 9:41 PM
To: [hidden email]
Subject: Re: mCherry and 2P

I've never tried this but looking at all the other replies my
guess would be that your IR blocking filter in the MP is blocking
the mCherry fluorescence. Check out what that filter is. I certainly
find that our blocking filter on our Leica SP2 MP blocks chlorophyll
fluorescence, for example. What is your blocking filter?

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [E]
Sent: Wednesday, 24 June 2009 4:26 AM
To: [hidden email]
Subject: mCherry and 2P

Dear Listers,

I am wondering if any of you have had success imaging mCherry in the 2-photon mode? We have mCherry bacteria rods that are very bright in confocal, but cannot get an image in 2P. We tried up to 1030nm (the coherent laser stops at 1040 and the power drops down to ~600mW at 1030), but with no success. Around 900nm we could detect a shadowy image of the bacteria, but at a power level that immediately bleaches the fluorophore.

Any suggestions?

Thanks,

Lily

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM

Internal Virus Database is out-of-date.
Checked by AVG.
Version: 7.5.560 / Virus Database: 270.12.26/2116 - Release Date: 15/05/2009 6:16 AM