Confocal Microscopy of Lymphocytes

[Ateh, Eugene]

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Hi All,
I am relatively new in confocal microscopy techniques and on this website. Can some please forward a suitable protocol for use on Lymphocytes. I specifically want to stain for cell surface markers cd 19, cd 20 cd 3 etc. I have the cells growing in culture but I just don't know how to proceed. I have downloaded some protocols but i get some conflicting ideas about how to properly handle lymphocytes.
Thanks for assistance.
Eugene
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Shivaprasad Bhuvanendran [[hidden email]]
Sent: Tuesday, December 14, 2010 12:16 PM
To: [hidden email]
Subject: Re: DsRed vs 2P

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Hi Staffan,

On the Zeiss LSM system that you are using, you can try an 'Excitation
Fingerprinting' macro to generate the excitation spectra of different
fluorochromes. The macro, available from Zeiss, automates the process of
recording the emission signal at different IR wavelengths at a pre-defined
step size while keeping the laser power constant. When manually changing the
wavelengths, you may not be able to accurately adjust the AOM settings to
compensate for change in laser power. Using this technique, you should be
able to determine the optimum excitation wavelength for the combination of
fluorochromes you have.

 We have successfully excited  DsRed (in combination with other
fluorochromes) at 910-940nm, without bleedthrough in the green
channel(bandpass 490-540), on a Olympus FV-1000MPE microscope using a
Coherent's Chameleon Vision laser .

In your case, as Ann has suggested, the variant of the DsRed that you are
using may also be a problem.

Best,
Shiva

Shivaprasad Bhuvanendran
Research Support Specialist - Bio-Imaging
The Rockefeller University
1230 York Avenue
Box 209 (DWB 201)
New York NY 10065
tel [phone]
fax [phone]

2010/12/14 Staffan Nyström <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> @Craig
>
> I have tried 10 nm steps from around 710 up to 1000 nm but to no great
> awail.
> I do get some signal out, but nowhere near the output I get with the 561
> laser.
> Since the emission of for example EGFP (at ~910nm) or DAPI (at ~750nm) is
> detected without problems (using NDDs) there shouldn't be any problems with
> blocking from filters (- or?)..
>
> Best Staffan
>
> --
>
> What wavelength did you have your Ti:Saph tuned to?  The 2p wavelength
> which
> a dye prefers is often different from what you would expect.  Try tuning
> the
> laser around its full range and see if you get better signal anywhere else.
>  Do you have any filters in your microscope that could be blocking the
> laser
> or causing other problems?
>
> Craig
>
>
> 2010/12/13 Staffan Nyström <[hidden email]>
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear list members,
> >
> > Has anyone any experience with visualizing DsRed using Ti:Saph (2
> photon)?
> > I have problems getting any decent fluorescence signal through between
> 700
> > and 1000nm excitation and there is significant bleedthrough
> > with EGFP. In the end I gave up since the "regular" 1 photon excitation
> way
> > gave a much better S/N ratio with minimal bleed through.
> >
> > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> > water dipping objectives.
> >
> > Best
> >
> > Staffan Nystrom
> >
> > PhD student
> >
> > MBB / Oncology & Pathology
> >
> > Karolinska Institutet
> > Stockholm, Sweden
> >
>

[mcammer]

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*****

Based on what you want to stain, I expect the protocols fall into two camps.  Each has benefits.

One probably is the labeling before fixation, usually with cells on ice.  This has benefits of maximum antigenicity and labeling only activity on the outer surface.  However, morphology is not ideal.

Fixing hard and fast before exposing to antibodies preserves morphology, but may impede antigenicity, may require autofluorescence  quenching steps and may permeabalize the cells.  Since you want to do confocal, most likely the morphology is more important and you should go with fixation first.


Sincerely,

Michael

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Ateh, Eugene [[hidden email]]
Sent: Tuesday, December 14, 2010 12:43 PM
To: [hidden email]
Subject: Confocal Microscopy of Lymphocytes

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi All,
I am relatively new in confocal microscopy techniques and on this website. Can some please forward a suitable protocol for use on Lymphocytes. I specifically want to stain for cell surface markers cd 19, cd 20 cd 3 etc. I have the cells growing in culture but I just don't know how to proceed. I have downloaded some protocols but i get some conflicting ideas about how to properly handle lymphocytes.
Thanks for assistance.
Eugene
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Shivaprasad Bhuvanendran [[hidden email]]
Sent: Tuesday, December 14, 2010 12:16 PM
To: [hidden email]
Subject: Re: DsRed vs 2P

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Staffan,

On the Zeiss LSM system that you are using, you can try an 'Excitation
Fingerprinting' macro to generate the excitation spectra of different
fluorochromes. The macro, available from Zeiss, automates the process of
recording the emission signal at different IR wavelengths at a pre-defined
step size while keeping the laser power constant. When manually changing the
wavelengths, you may not be able to accurately adjust the AOM settings to
compensate for change in laser power. Using this technique, you should be
able to determine the optimum excitation wavelength for the combination of
fluorochromes you have.

 We have successfully excited  DsRed (in combination with other
fluorochromes) at 910-940nm, without bleedthrough in the green
channel(bandpass 490-540), on a Olympus FV-1000MPE microscope using a
Coherent's Chameleon Vision laser .

In your case, as Ann has suggested, the variant of the DsRed that you are
using may also be a problem.

Best,
Shiva

Shivaprasad Bhuvanendran
Research Support Specialist - Bio-Imaging
The Rockefeller University
1230 York Avenue
Box 209 (DWB 201)
New York NY 10065
tel [phone]
fax [phone]

2010/12/14 Staffan Nyström <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> @Craig
>
> I have tried 10 nm steps from around 710 up to 1000 nm but to no great
> awail.
> I do get some signal out, but nowhere near the output I get with the 561
> laser.
> Since the emission of for example EGFP (at ~910nm) or DAPI (at ~750nm) is
> detected without problems (using NDDs) there shouldn't be any problems with
> blocking from filters (- or?)..
>
> Best Staffan
>
> --
>
> What wavelength did you have your Ti:Saph tuned to?  The 2p wavelength
> which
> a dye prefers is often different from what you would expect.  Try tuning
> the
> laser around its full range and see if you get better signal anywhere else.
>  Do you have any filters in your microscope that could be blocking the
> laser
> or causing other problems?
>
> Craig
>
>
> 2010/12/13 Staffan Nyström <[hidden email]>
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear list members,
> >
> > Has anyone any experience with visualizing DsRed using Ti:Saph (2
> photon)?
> > I have problems getting any decent fluorescence signal through between
> 700
> > and 1000nm excitation and there is significant bleedthrough
> > with EGFP. In the end I gave up since the "regular" 1 photon excitation
> way
> > gave a much better S/N ratio with minimal bleed through.
> >
> > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> > water dipping objectives.
> >
> > Best
> >
> > Staffan Nystrom
> >
> > PhD student
> >
> > MBB / Oncology & Pathology
> >
> > Karolinska Institutet
> > Stockholm, Sweden
> >
>

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-----Original Message-----
From: "Ateh, Eugene" <[hidden email]>
Sender: Confocal Microscopy List <[hidden email]>
Date:         Tue, 14 Dec 2010 12:43:40
To: <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Subject: Confocal Microscopy of Lymphocytes

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi All,
I am relatively new in confocal microscopy techniques and on this website. Can some please forward a suitable protocol for use on Lymphocytes. I specifically want to stain for cell surface markers cd 19, cd 20 cd 3 etc. I have the cells growing in culture but I just don't know how to proceed. I have downloaded some protocols but i get some conflicting ideas about how to properly handle lymphocytes.
Thanks for assistance.
Eugene
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Shivaprasad Bhuvanendran [[hidden email]]
Sent: Tuesday, December 14, 2010 12:16 PM
To: [hidden email]
Subject: Re: DsRed vs 2P

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Staffan,

On the Zeiss LSM system that you are using, you can try an 'Excitation
Fingerprinting' macro to generate the excitation spectra of different
fluorochromes. The macro, available from Zeiss, automates the process of
recording the emission signal at different IR wavelengths at a pre-defined
step size while keeping the laser power constant. When manually changing the
wavelengths, you may not be able to accurately adjust the AOM settings to
compensate for change in laser power. Using this technique, you should be
able to determine the optimum excitation wavelength for the combination of
fluorochromes you have.

 We have successfully excited  DsRed (in combination with other
fluorochromes) at 910-940nm, without bleedthrough in the green
channel(bandpass 490-540), on a Olympus FV-1000MPE microscope using a
Coherent's Chameleon Vision laser .

In your case, as Ann has suggested, the variant of the DsRed that you are
using may also be a problem.

Best,
Shiva

Shivaprasad Bhuvanendran
Research Support Specialist - Bio-Imaging
The Rockefeller University
1230 York Avenue
Box 209 (DWB 201)
New York NY 10065
tel [phone]
fax [phone]

2010/12/14 Staffan Nyström <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> @Craig
>
> I have tried 10 nm steps from around 710 up to 1000 nm but to no great
> awail.
> I do get some signal out, but nowhere near the output I get with the 561
> laser.
> Since the emission of for example EGFP (at ~910nm) or DAPI (at ~750nm) is
> detected without problems (using NDDs) there shouldn't be any problems with
> blocking from filters (- or?)..
>
> Best Staffan
>
> --
>
> What wavelength did you have your Ti:Saph tuned to?  The 2p wavelength
> which
> a dye prefers is often different from what you would expect.  Try tuning
> the
> laser around its full range and see if you get better signal anywhere else.
>  Do you have any filters in your microscope that could be blocking the
> laser
> or causing other problems?
>
> Craig
>
>
> 2010/12/13 Staffan Nyström <[hidden email]>
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear list members,
> >
> > Has anyone any experience with visualizing DsRed using Ti:Saph (2
> photon)?
> > I have problems getting any decent fluorescence signal through between
> 700
> > and 1000nm excitation and there is significant bleedthrough
> > with EGFP. In the end I gave up since the "regular" 1 photon excitation
> way
> > gave a much better S/N ratio with minimal bleed through.
> >
> > Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA
> > water dipping objectives.
> >
> > Best
> >
> > Staffan Nystrom
> >
> > PhD student
> >
> > MBB / Oncology & Pathology
> >
> > Karolinska Institutet
> > Stockholm, Sweden
> >
>