Posted by
Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5839005.html
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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 <
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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
(510) 643-9603
(510) 643-9500 fax
2010/12/13 Staffan Nyström <
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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
>
=