My experience is that laser light (at the intensities required for
     most fluorescent imaging) is not more damaging to live cells than
     the mercury lamp. Indeed, I have observed much faster
     photobleaching and concommittant photodamage from standard
     epifluorescence than with confocal. However, I cannot put any
     absolute numbers on this.

     Anna Smallcombe PhD
     Bio-Rad UK


______________________________ Reply Separator _________________________________
Subject: multiuser facility decision
Author:  Confocal Microscopy List <[log in to unmask]> at
Internet
Date:    26-11-2001 1:09 pm


Hi,
I am in the process of making the decision regarding which system to
purchase for a multiuser physiology/imaging facility. The major users of
this instrument have the following needs:

1)combine patch clamp recording with imaging chloride levels using MEQ
(excitation 330, emission 450) and/or calcium levels (Fura preferred)
2) combine patch clamp recording with analysis of cytoskeletal alterations
associated with channel activity - combination of GFP and 2 antibody labels
- timescale in the order of 100s of msec
3) timelapse analysis of growth cone outgrowth in cultured cells with gfp
and 2-3 antibody labels (imaging over minutes to hours)

We'd also like the flexibility of imaging axon outgrowth (DiI label) in
150-200 um slices over a 24 hour period, but realize this is probably not
attainable given our other needs and budget (260K easy, more theoretically
possible, but...)


Our first choice is the DeltaVision deconvolution/restoration system because
of it's outstanding 3 dimensional ultrastructural resolution, the flexibilty
and live tissue compatibility of a mercury lamp compared with a laser
approach, and a software package for acquistion, analysis and display that
meets most of our needs. However, we are now also considering purchase of a
confocal instrument because of difficulties combining acquisition of
physiological data with imaging on the DeltaVision (no software for
synchronizing physiology and imaging equipment, cumbersome approach to
attaching manipulators for patch clamp electrodes to the motorized stage,
unknown electrical noise associated with the multiple motors of the system).
I discussed most of the current fast acquisition confocal instruments with
vendors at the Society for Neuroscience meeting earlier this month and must
confess to being slightly overwhelmed by the multitude of choices. I welcome
any advice, but am specifically looking for the answers to the following
questions:

1) Is anyone using MEQ with a confocal system? Which laser?

2) Has anyone attempted to make physiological recording measurements in
conjunction with the DeltaVision?

3) How compatible are the current crop of confocal instruments with live
tissue imaging multiple times?

4) Does anyone have experience with the Olympus FV500 in a combined
phsyiology/imaging setup? Is the experience positive?

5) Does anyone have experience with the Solamere spinning disk confocal in
a combined physiology/imaging setup? Is the experience positive?

6) Though it's probably out of our budget range, how well does the Zeiss
510 Meta really work for simultaneously acquiring multiple fluorochrome
information and then separating them by analysis of emission spectra (in 4
dimensions including time)?

7) Has anyone had difficulty with any particular system in synchronizing
physiological recordings with image acquisition?

The vendor specific questions above are not meant to imply we have narrowed
the search to only those instruments. If you would like to provide vendor
specific responses you think might be inappropriate for public display,
please email me directly, rather than the list, or call me at 802 656 8060.

Thank you,
Cindy Forehand

Dr. Cindy Forehand
Department of Anatomy and Neurobiology
426A Health Science Research Facility
University of Vermont
149 Beaumont Avenue
Burlington, VT 05405-0075
(802) 656-8060
[log in to unmask]






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