http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5839979.html
you tune the less sensitive the pulse is to dispersion. This may account
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>
> Remember that the pulsewidth will also be changing as you tune, so if you
> have
> a pulse compressor, you may want to tweak that too to keep it constant.
> Though
> the dependence for 2P is linear, pulsewidths can change quite drastically
> towards the ends of the tuning range of a Ti:sapph.
> Sudipta
> On Wed, 15 Dec 2010 06:38:48 -0500, Andreas Bruckbauer wrote
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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
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
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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]>
> >
> > > *****
> >
> > > To join, leave or search the confocal microscopy listserv, go to:
> >
> > >
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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
> >
> > >
> >
> > =
>
>
> Dr. Sudipta Maiti
> Associate Professor
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: 91-22-2280-4610
> alternate e-mail:
[hidden email]
> url: biophotonics.wetpaint.com
>