>The tube gain is also a nonlinear function of applied voltage, so you may have
>similar ultimate sensitivity, even if at low gain one PMT needs a higher bias
>voltage. Therefore, it does not follow that you can reduce the gain by a factor
>of two with an RSI of 2. The real question is whether your signal to noise
>ratio improved.
>
Ok. So let me turn this into a more biological problem. You're imaging a live cell (which can be damaged by laser irradiation) with a constant level of fluorescence. You need an optical section so you're not going to open the pinhole. So ideally what you want is a detector which allows you to pick up a similar signal (with a better signal-to-noise ratio) but with a lower laser power. In this case, you have to depend on your gain (or voltage applied to the PMT) to adjust the intensity of the signal. Shouldn't then the sensitivity of the PMT be related with the gain settings ?



>Concerning accumulate to peak, the number of frames required depends a lot on
>the kind of noise you have. Are the images obtained after 2 passes with the old
>PMT are as good as the images obtained after 6 passes with the new PMT?

Well, the test performed is with no sample, just using the brightfield lamp of the scope as the constant light source. So it's difficult to say what is good and bad. Indeed, the histogram standard deviation is higher in the image collected after 2 passes.

>The accumulate-to-peak function would be much more useful if >it had smarter end conditions. For example, one could >require that a minimum percentage of pixels reach a certain threshold.

In this case I must say that we only followed the Bio-Rad protocol. We did several other tests like imaging different
samples and 200 nm fluorescent beads. But here was just to test and compare different settings on both new and old PMTs. And in these tests, the image collected by the "enhanced" PMTs was always extremely similar to the one we had.


 Rui





Rui Malho'
__________________________________________
Dept Biologia Vegetal, FCL, Bloco C2
Univ de Lisboa, Campo Grande, 1780 Lisboa,
Portugal. Tel. 01 7500069  Fax. 01 7500048
Email: [log in to unmask]