Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

George McNamara wrote:

> The Leica approach is nice for setting "virtual bandpass", but is not a 
> practical approach for spectral scanning - any real fluorophores will 
> likely be dead long before you reach the end of a high resolution 
> spectral scan. 

That is certainly not my experience.  If your sample will handle a
big Z-series it should handle a spectrum.  From the point of view
of the fluorochrome there's not much difference.  I'd usually do the
spectral scan at 256*256 since in the end you don't usually need
all that much spatial resolution.

Anyone who was at our recent workshop on photoactivatable fluorescent
will have seen our Leica SP2 do spectral scans on a sample on which
a ROI had been converted - with no visible bleaching or further
conversion (though sometimes the bacteria moved!).  I wasn't
quite running at Nyquist but still using many more points than
a 32-channel system.  And the spectra showed the conversion very
clearly.

  > Guy Cox's (10/29/2006 spectrofluorometer listserv message) of using an
> Ocean Optics spectrometer, is starting to sound more and more appealing. 
> Just get a spectrum. Could stage scan if you really wanted an image - do 
> any of the commercial CLSM's even have stage scanning options?

It's certainly a cheap option and gives a good spectrum, but you'd
need to change your illumination to give a point if you really want
pixel by pixel spectra.  And then the s/n wouldn't be good unless
you went just as slowly as the SP2.  And a scanning stage isn't cheap.
With conventional epi-fluorescence you can close the field iris to
isolate an area of interest which is often what you really want anyway.
If you have two c-mounts you can record a wide-field image as well as a
spectrum.

> It looks to me that, in the current literature, the best way to do FRET 
> imaging is with FLIM, for example, Vogel's new Biophysical J article 
> (Koushik et al, 
> http://www.biophysj.org/cgi/content/abstract/biophysj.106.096206v1 
> ,contact Steve for the supplemental file since it is not currently 
> online). He uses the multiphoton laser as the light source and a high NA 
> condenser with the Becker&Hickl FLIM unit on the condenser side. The 
> only downside of his way is that the FLIM unit is about $70K-$100K, not 
> counting the ~$170K for a current generation Ti:Sapphire.

But pulsed diode lasers are perfectly adequate and FAR cheaper.
I've used Picoquant lasers with both Becker & Hickl and Nikon LIMO
systems.  The LIMO is much cheaper than the B&H too.  The time is
also an issue with the B&H - lifetime resolution is fantastic, but
to get that you're collecting just as long as a spectral scan on
the Leica.  The LIMO is much quicker (but lower res). Once again,
it's horses for courses!

         						Guy
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net