Posted by
Staffan Nyström on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5835535.html
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@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
>