Dear James,

thanks for your email. Once again I'd like to point out that much of the credit
should go to Lauran Oomen, who really measured it very meticulously.
Indeed, it may be doable to separate individual laserlines and monitor them on
a pixel-by-pixel base. If this would work, it would probably be the single best
solution. But it involves quite some optics, at the expense of flexibility. If
for instance I would choose to have another laserline installed, I would need
to change filters for the relevant diode detector as well.

Yes, we can rule out vibration and dust. Vibrations are expected to have the
same frequency for all laser lines. Dust could may be be responsible for
step-like changes, but hardly for oscillatory behaviour.

Regards, Kees

James Pawley wrote:

>
> Kees,
> Thank you for bringing this important matter up and for organizing the
> responses for us.
>
> I think that you may be giving up a little two quickly on normalizing the
> stored data, based on the output of laser-power sensors in the scan-box.
> There could be one sensor for each line and each could have a filter on it
> so that it only sensed the correct light. (like the 3-channel Bio-Rad
> Transmission Filter).  Assuming that about 10% of the fiber output was
> diverted to the sensor (by a piece of flat glass at 45 deg to the beam
> sending the light to a diffuser), the signal levels would be about 100,000x
> larger than the fluorescent signal and therefore easy to sense with simple
> a photodiode.
>
> One wouldn't store the sensed data but immediately use it to digitally
> normalize the output from the fluorescence detector and then store the
> normalized result.
>
> This isn't something that owners could do for themselves but at least it
> offers hope for the future.
>
> It is interesting to note that several of the original confocals
> (particularly that designed by Kjell Carlsson) used diodes to monitor and
> correct for laser instability. They disappeared probably because, in the
> age before fiber-optic light delivery, it was easier to stabilize the laser
> output directly.  Now we see that single-mode fibers have added a new
> instability.
>
> I assume that you can rule out vibration and dust as variables? If the
> laser changes its pointing direction for any reason, this will change the
> amount of light focussed into the fibre. And a dust particle landing near
> the fibre entrance has the same effect.
>
> Cheers,
>
> Jim P.
>
>               ****************************************
> Prof. James B. Pawley,    (on Sabbatical)       Ph.  61-2-9351-7548/2351
> Room LG 10, Madsen Building, F-09,              FAX  61-2-9351-7682
> University of Sydney, NSW, 2006 Australia       [log in to unmask]
> "A scientist is not one who can answer questions but one who can question
> answers."       Theodore Schick Jr., Skeptical Enquirer, 21-2:39

--

Kees Jalink Ph.D.
The Netherlands Cancer Institute, dept. of Cell Biology H1
Plesmanlaan 121, 1066CX Amsterdam, the Netherlands
020-5121933 (tel)   / 020-5121944 (fax) / [log in to unmask] (email)
at home: Paulus Potterlaan 5, 2102CC Heemstede
0235-476047 / [log in to unmask]