Hi Joseph,
You'd probably have more joy if you could take the visible light out of the
beam, and use UV only. The calcium-free indo fluorescence can usefully be
picked up between 470 and 550 nm, I use a 530 df 20 (fluorescein filter)to
discriminate it, and reduce the signal from 488 scatter from my primary
laser, if I didn't have any 488 laser light kicking around I'd probably use
a 490 nm filter. In your case you'll have 488 and presumably 514 side
scatter co-linear with indo fluorescence, and you'd find it hard keeping
the scatter from contributing an offset to your blue/green indo signal.
If you do settle on UV only excitation, remember to change or remove the
filters in front of your FSC and SSC detectors so that the UV can get
through.
If you use a dichroic to split the laser for the above reason, you'll be
able to get separate power readings for both beams.
Ray
At 14:06 +1100 8/1/98, Joseph Webster wrote:
>Me again!
>I need to work up calcium measurements on the FACStar Plus, and exploring
>the options.
>
>Since we have the right laser and mirrors, I wish to run the primary laser
>in multi line UV+VIS, so I can get the usual scatter along with calcium
>ratio in FL1 & FL2.
>We could also use the dye laser for other markers at the same time.
>
>While I'm waiting for the optical filters to arrive:-
>Has anyone tried this?
>Are there glaring reasons why it should not work?
>Any ideas for measuring the power of 488 & UV separately in multi line?
> (yes, I have a power meter)
>
>I know there are many people using other laser layouts, but we only have
>a two-laser bench and I REALLY don't want to move the dye laser in and
>out of position between experiments.
>
> Many Thanks, Joseph.
>
>--
>Joseph Webster
>Flow Cytometry Facility
>Centenary Institute
Ray Hicks
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