Posted by
Paul Herzmark on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5836745.html
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Using my Tai-Saphire laser, I measured the optimal 2P excitation wavelength
for TD Tomato (a different red fluorescent protein).
Since the wattage coming out of the laser varies with wavelength you need to
correct for that. I have a pickoff after my laser power attenuator that goes
to a power meter so I can keep the power delivered to the cells constant.
Scan through the wavelengths changing the attenuator to keep the power
constant and look for the brightest emission. I found 1040 nm (the end of
what my laser will deliver) to be about 5X more efficient per watt than 920
nm.
It was still going up when I got to 1040 so I am going to try an OPO
(Optical Parametric Oscillator) to look at even longer wavelengths.
Keeping the delivered power constant and scanning the spectrum is also a
good way to find the optimum compromise power for simultaneously exciting
several fluorophores.
Paul
Paul Herzmark
Specialist
[hidden email]
Department of Molecular and Cell Biology
479 Life Science Addition
University of California, Berkeley
Berkeley, CA 94720-3200
(510) 643-9603
(510) 643-9500 fax
2010/12/13 Staffan Nyström <
[hidden email]>
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>
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>
> 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
>