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Rather than matching the laser power at the sample, I think it would be
better to match the S/N in the images.

Cheers,
Jeff Reece
Biomedical Engineer
Confocal Microscopy Center
National Institute of Environmental Health Sciences (NIEHS)
P.O. Box 12233, MD F2-02
111 Alexander Drive, Bldg. 101 / Rm. F219
Research Triangle Park, NC  27709  USA
(919) 541-0311
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-----Original Message-----
From: tony collins (BI) [mailto:[log in to unmask]]
Sent: Wednesday, April 02, 2003 10:43 AM
To: [log in to unmask]
Subject: disk vs point scanning bleach rates


Search the CONFOCAL archive at
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Hi Folks

I've some data regarding the apparent lower-bleach rates of fluorophores
associated with disk-based compared to conventional scanning confocals. I'd
appreciate some feedback regarding the validity of the approach.

I've looked at the bleach rates for calcein-AM loaded (1 um for 20min + 30
min de-estrification) and YFP-IP3R1 expressing cells (it's what I had left
over...) on a Biorad MRC1024ES and PE Ultraview confocal. Both confocals are
on Nikon "Eclipse" microscopes, both with x60, 1.4NA, PlanApo objectives.
Both with 488 nm laser intensity measured as 60 uW at the objective with a
coherent Lasercheck power meter (this measurement is my main query - is this
a valid way to balance the intensities?). Both driven at approx. one 512x512
frame per second. Both with pixel size approx 0.12 um. Continuous
illumination for both. Is there any other relevant info I've left out?

Data was fitted a single exponential decay giving the following half-times
for bleaching:

Perkin Elmer Ultraview
Calcein:  77 sec                YFP: 232 sec

Biorad MRC1024
Calcein:        11 sec  YFP:    22 sec

This is preliminary data and I was wanting some feedback regarding this
approach before I firm it up.

I adjusted the zoom on the Biorad to give a similar pixels size to the PE
system. Was this correct?

Another concern is the way in which the two systems deliver excitation light
to the specimen and the way in which the power meter measures intensity may
confound the use of this power meter. Am I being unduly cautious? Can I use
this power meter to compare excitation intensities between disk and
point-scanning confocals? Is it a "good enough" measure? If not, is there a
way that this can be done simply so that the bleach rates obtained at the
end of the day say something about the confocals, not the powermeter!?

Any suggestions much appreciated.

Trawling through the archive I found reference by JimP to some work by Eric
Manders on multi-beam vs single beam damage. Has there been any follow up?

Thanks

Tony

PS. Apologies to Biorad, but I'm in the process of generating PSFs for the
two systems so you'll have your day soon!

Tony Collins Ph.D.
Senior Research Associate
Calcium Group
Laboratory of Molecular Signalling
Babraham Institute
Cambridge, UK, CB2 4AT
Tel: +44 (0)1223 496499
Fax: +44 (0)1223 496043

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