http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5839142.html
a pulse compressor, you may want to tweak that too to keep it constant. Though
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>
> The pick up from the laser is a good idea and i think microscope
> manufacturers should implement this and get rid of the percentage
> numbers for laser powers which are widely used. However this will
> not account for the differences in transmission of the different
> optical elements in the microscope. So the Zeiss macro is a good
> idea if you are interested in the fluorophore characteristics. The
> only thing to keep in mind is that, if your sample allows you to use
> more power and you are operating at 100%, you still might gain from
> using the lower wavelength. The fluorescence goes with the square of
> the laser power, so 2.5x more power translates to 6x more
> fluorescence, if the fluorescent protein is 3x more efficient at the
> longer wavelength (at same power level), you would still get 2x more
> fluorescence using the lower wavelength at higher power. But these
> are just theoretical values.
>
> best wishes
>
> Andreas
>
> -----Original Message-----
> From: Paul Herzmark <
[hidden email]>
> To:
[hidden email]
> Sent: Tue, 14 Dec 2010 22:20
> Subject: Re: DsRed vs 2P
>
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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
>
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>
> (510) 643-9500 fax
>
> 2010/12/13 Staffan Nyström <
[hidden email]>
>
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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
Dr. Sudipta Maiti
Dept. of Chemical Sciences
Ph. 91-22-2278-2716 / 2539