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Hi Kathy -

I think the best way to see if your fluorophores are saturated is just 
to do the experiment.  If you have some way of attenuating your light 
source (ND filter is best, but you could probably just close down the 
aperture diaphragm) you can plot fluorescence intensity vs. excitation 
intensity and see if it saturates or not.

My experience with conventional arc lamps in widefield microscopy is 
that the light intensities are at least 20-fold below saturation for 
dyes like GFP so I would guess that you're unlikely to be at saturation 
unless the microlenses are really good at increasing the excitation 
power at the illumination point.  But I think you're best off doing the 
experiment.

Kurt

Kathryn Spencer wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello;
> 	Should have supplied more information. Both illumination sources
> were tested on spinning disk systems (one with microlenses, one
> without). Appropriate filters were in place for the metal halide source,
> and were compared to specific laser lines (diode or ion). We took the
> objective out of the system, just to eliminate differences between NAs.
> I guess my basic question; is more always better? If you have short
> exposure times (200msec) with the lower intensity illumination source,
> what does 10 times that really get you? Once the fluor is saturated, it
> cannot absorb any more photons. Am I at that saturation point?
> 	The power meter is a broadband power/energy meter that does not
> need tuning to specific wavelengths. It has a thermopile sensor.
> 	We're demoing two systems, and my colleagues are excited (pun
> intended) about the one system, because it is "10 times better". Is it
> really?
> 	Kathy
>
>
>
>
>   


-- 
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

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