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Hi Mark

Yes, I am looking for non-linear behaviour between illumination intensity and bleaching rate in the two. This was more out of academic interest rather than as a real world comparison between confocal systems where S/N would be far more relevant in determining confocal performance.

Apologies, I didn't make this clear and it's why I haven't included any info about the detector. I was trying to get at whether the lower bleach rate many of us see with the disk based system is due to either lower laser intensity being used to give similar S/N (i.e. it's due to the detector) or whether the lower bleach rate is due to non-linear bleaching and is an intrinsic property of the disk system.

The power meter was held at the objective whilst scanning. I'm assuming this means that it was a measure of average power while scanning. I tried to match up the field of view "exposure" by running both systems at about 1 frame per second (i.e. quick for the biorad, slow for the PE system!). I'm hoping this means that the "pixel exposures" per frame are similar, but for the biorad the exposure comes all at once and for the PE system the exposure "flickers".

I hope this makes sense and is correct!

Tony



> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Did you measure average power while scanning? It's just that
> if one exposes the
> sample for longer per acquired frame it might not show up in
> the numbers you
> gave. I assume you are looking for non-linear behavior
> between illumination
> intensity and bleaching rate in the two?
>
> Mark
>
> "Reece, Jeffrey (NIH/NIEHS)" wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > 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
> > [log in to unmask]
> >
> > -----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
> > 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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