>
> I NEVER use the HeNe laser for confocal fluorescence imaging. I am running a
> Zeiss LSM410 with 543 HeNe laser + 488/568/647 ArKr laser. Why would you use
> a weak "peak mismatched" 543 laser to excite green or red fluos when you
          ^^^^^^^^^^^^^^^
Organic fluorophores, especially in solution, tend to have broad emission
spectra. We often excite FITC at 514nm at 10% power (which means about
1.5mW out of the laser) simply to reduce the FITC emission relative to
a second dye (eg Cy3 -- which we also excite at 514nm). Ok -- our set up
is "laser challenged" (ie we only have the argon laser on the MRC-1024).
But the point is that you can get away with being far from the official
excitation peak. I say "official" peak because  the
excitation and emission peaks can be all over the place depending on the
environment (mounting medium, aqueous, presence of lipid, detergents, etc).
We have also had good results with Cy5 exciting at 514nm,
though it is obviously not deal.

Having said that -- it is certainly nice to have a large selection of lines
to work with, when you can afford it. If the cost of an Ar/Kr laser
(both initial cost and shorter tube life) is not of concern, I am sure that
the 568nm line can come in handy.

While we are talking about lasers, has anyone equipped a UV confocal with a
354nm HeCd laser (most such lasers are 325nm or 442nm, but there is one
manufacturer who makes a 354nm HeCd). It should be cheaper than the argon-ion
UV laser and also is air cooled (the UV argon ion is generally a multiwatt
affair that needs serious water cooling). How about a tripled Nd:YAG?

--aryeh
Aryeh Weiss                          | email: [log in to unmask]
Department of Electronics            | URL:   http://optics.jct.ac.il/~aryeh
Jerusalem College of Technology      | phone: 972-2-6751146
POB 16031                            | FAX:   972-2-6751275
Jerusalem, Israel                    | ham radio: 4X1PB/KA1PB