> Hi All,
> I am trying to decide between a 543 HeNe line or 568 Kr laser for the
> green line for a confocal system beside the longer life of the HeNe
> and the better seperation from the 488 with the Kr are there any
> other factors I should take into account?
>

Dear Khaled,

We have just installed a new TCS-SP confocal with Argon 488, HeNe 543
and Hene 633 lasers for the sake of reduced maintenance costs and
"longevity" - (too many bad experiences with ArKr's).   The most
powerful  543HeNe available to fit into the confocal was a 1.5mW,
which delivers way <1mW to the specimen, compared to the ±10mW for
the 568Kr laser which delivers several mW to the specimen. Despite
the reduced power available, we have not experienced any
problems imaging PI, Cy3, Tritc. There are also enough new
protein labelling dyes around the 540nm mark so you
won't miss Texas Red (Alexa546, Cy3, BODIP TMR etc).

Since your "red" channel will now be 10nm closer to
the green channel, apart from changing beam splitters, you should
probably throw away regular fluorescein (in case you haven't
already), and switch to dyes with narrower emission spectra (Oregon
green, BODIPY488, or Alexa488 etc).

There are also plenty of dyes in the 540nm excitation range for
living cells so there should be no problem.

Cheers,

Ian.


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Dr Ian S. Harper
Confocal Microscopy Facility
Department of Biological Sciences
Monash University, Clayton, Vic 3168, Australia
Email: [log in to unmask]
Tel: +61 3 9905 5635;  Fax: +61 3 9905 5613
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