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Hi Tony,
What an interesting experiment!
One additional important variable of importance is the frame size.
How many pixels in the raster?
If your disk scanner puts its 60 microwatts over a larger area than
you scan with the Biorad, the dose/pixel (you say the pixels are the
same size) may not be the same in each case.
In addition, most 1024's don't have the ability to blank the beam
when it is not actually collecting data (i.e., on he retrace of each
line). If this is the case, then the dose just outside the field of
view on either side will be huge (because the beam slows down to turn
around) compared to that in the imaged area. If your cell has a
process that extends into this high dose are, it might be
preferentially damaged.
How did the images look? You don't mention the type of CCD camera but
I would expect that the performance of the disk system will greatly
improve if you can manage to acquire well-designed camera using the
E2V gain Register chip with an effectine read noise of less than one
electron/pixel/frame.
Finally, you should talk to Eric Manders (his email is above) who
has also performed comparisons of this type.
Keep up the good work.
JimP.
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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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--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
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"A scientist is not one who can answer questions but one who can
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