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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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