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Dear Confocalist,

OK, my turn.

If one is using an oil 1.4 NA lens, and are viewing within about 10
um of a coverslip surface, and are using a fluorophore that has an
extinction coefficient in the 60-80 k range or higher at 543 nm, then
100 uW will show some effects of saturation. However, looking deeper
into a sample will start increasing the effects of spherical
aberration and RI mismatch decreasing the power density within the
focal "volume,". Thus, the same small feature that looks fine at 10
um deep, starts to become much dimmer at 20 to 30 um deep. Of course,
there are tricks like opening the pinhole and performing
deconvolution on your confocal stacks.

Then there is the problem of using your favorite 20x 0.7 NA dry
objective. Now the power delivered per unit volume is reduced
somewhere between the cube or fourth root.

I guess what I am trying to say is that the use of a 1 mW laser, will
be very dependent on sample, and specific application and it would be
a good idea to elaborate on what applications people fine the HeNe543
useful or not.

My Best to Everyone,
Mario



>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi All,
>
>A couple of comments on recent postings on this topic:
>
>I couldn't agree more that the Green HeNe maximum power output is marginal
>in some usage applications, these most likely being live cell imaging with
>weak fluorescence and/or low efficiency emission/detection pathways. On
>fixed specimens, using appropriate fluorophores with efficient detection
>this should not be a problem at all, especially if one is prepared to
>integrate the signal for a few scans. As Jim Pawley pointed out, 100
>microwatts at the sample is likely to be saturating the fluorophore anyway.
>Are the problems that people are experiencing with the green 543nm HeNe
>laser due more to inefficiencies on the detection side rather than on the
>excitation side?
>
....


--
_________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies LLC
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