Posted by
Shivaprasad Bhuvanendran-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5836187.html
*****
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 +1 212 327 7487
fax +1 212 327 7489
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
> >
>