Sorry, I didnt realize Harvey had sent the note to the list. I told him that
I didnt mention it because it was old news. See the IMR's web pages for 2 and
3-label images:
http://www.bocklabs.wisc.edu/imr/facility/2p.htm
or the paper:
Wokosin, D. L., Centonze, V. E., White, J. G., Hird, S. N., Sepsenwol, S.,
Malcolm, G. P. A., Maker, G. T. and Ferguson, A. I. (1996). Multiple-photon
excitation imaging with an all-solid-state laser. Proc. of Optical Diagnostics
of Living Cells and Biofluids, San Jose, SPIE.
I myself have done nice 2-label imaging, using DiO and DiA, both normally
excited by blue light. I used 850 nm. Yes there is
emission crosstalk, identical to 1-photon, but this can be removed
computationally, as long as
you have some part of the image with each label isolated, or a sample to tell
you what the crosstalk ratios are. You probably cant get 3-photon excitation of
Hoescht with a Ti:Sapph, only 2-p. Wokosin used 1047 nm Nd:YLF laser to get 2-p
excit. of rhodamine and fluorescein, and 3-p of Dapi.
And to his other Q I suggested he read a small paper I wrote in the Dec 96 issue
of Biology, "Vital imaging: Two photons are better than one." Curr. Biol. 6:
1595-1598, for an assessment of pros and cons of multiphoton and confocal.
Basically, cost and possible absorption of IR by some pigmented cells are the
only downs of 2-photon.
Sorry, I am out of reprints. Scott Fraser may still have a couple.
Hope this helps!
Steve Potter
In message <[log in to unmask]>
Confocal Microscopy List writes:
> I am interested a reply to this also. Multi-photon acquisition is
> impressive in its live imaging capabilities and for the depth
> it can penetrate into a sample. I haven't seen good
> acquisition of double, much less triple labelled samples. We ended up
> collecting GFP and Texas red with the krypton-argon laser and Hoechst with
> the Ti-sapphire.
>
> Paulette Brunner
> W.M.Keck Center for Advanced Studies in Neural Signaling
> University of Washington
> Seattle, WA 98195-7290
>
> (206) 685-8784
> (206) 685-0619 (fax)
>
> [log in to unmask]
>
> On Wed, 20 Aug 1997, Harvey J. Karten, M.D. wrote:
>
> > Steve,
> > Nice summary of the recent multi-photon workshop. You didn't
> > mention
> > anything about the feasibility to do double/triple labeling of
> > fluorophores witgh a 2/3 photon system? I know that you could separate
> > the emission spectra, but can you reliably generate single fluorophorer
> > excitation with this system? I was much intrigued by your comment that
> > you are ready to pitch the confocal in favor of the multi-photon, but
> > you made not comment about any remaining virtues of the standard
> > confocal. Can you comment?
> >
> > --
> > Harvey J. Karten, M.D.
> > Dept. of Neurosciences
> > University of California @ San Diego
> > La Jolla, CA 92093-0608
> > WCBR EMail: [log in to unmask]
> > Other EMail: [log in to unmask]
> > Phone (Lab): 619-534-4938
> > FAX (Lab): 619-534-6602
> > Home Phone: 619-755-8573
> > Retina Information System: http:/www-cajal.ucsd.edu
> >
______________________________________________________________
Steve Potter, Philosophical Doctor
Pine/Fraser Labs
Caltech Division of Biology 156-29
Pasadena, CA 91125
[log in to unmask]
http://www.caltech.edu/~pinelab/pinelab.html
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