On Thu, 15 Sep 1994, Ardan Patwardhan wrote:
 
> I would like to thank all the people who replied
> to my inquiry for their help and input.
>
> If I understand correctly, triple excitation confocal
> microscopy is presently done by using a Argon/Krypton
> multiline laser and then selecting a wavelength by using
> a filter wheel placed infront of the laser. This would
> mean that one first scans a stack of images using one
> excitation wavelength, then the next and then the next.
> This would mean that the translation stage  in the z
> direction would have to move up/down 5 times in order
> to scan in all three colors.
 
        In collecting triple labelled images, one would normally remain
in a fixed Z position until all three signals had been collected, and
then move on to the next z plane.  Alignment is therefore not a problem
(especially if your system collects all three signals simultaneously)
>
> Another question I have is concerning the relative intensities
> of the three beams. With a multiline laser, one usually has a
> fixed relation between the output power at one wavelength and
> the output power at another wavelength. Different fluorophores
> however have different fluorescent efficiencies. Is this a
> problem ?
>
No. You can quickly scan through your sample, and determine the optimum
gain and offset for your PMT's prior to taking high resolution images in
series.   In addition, software is available which allows you to make
adjustments to the brightness and contrast of your image once it is
collected (this includes z-series images).  However, it is far better to
have collect the "perfect" images in the first place.
 
 
Kem Rogers
University of Western Ontario