Dan Boorstein <[log in to unmask]> wrote
> Hello, our laboratory is in the market for a confocal imaging system,
> and we are looking for any input available concerning materials and/or methods.
> Our objective is to perform measurements of free cytosolic calcium
> concentration (ccc) in an intact mouse extensor digitorum longus muscle (10
> mg; 12 mm long x1.5 mm thick) mounted in a superfused preparation on a
> microscope stage.
> We feel that confocal capability is required because there is
> heterogeneity between cells in free ccc (i.e., cells above or below the cell
> in the focal plane do not have the same ccc); in addition, there is free ccc
> heterogeneity even within the same cell. Optical section thickness is not
> critical, up to 5 microns is acceptable.
> We will be using a ratioing dye, either Fura or Indo, because the
> measurements will be made over a long period of time (i.e., hours). Non
> ratioing dyes such as Fluo-3 would be lost from the cell over such a period
> of time and a decrease in fluorescence would not necessarily be attributable
> to a decrease in ccc.
**************
This is not necessarily the case, Dan: I have undertaken
experiments lasting over 48 hours (after loading with Fluo 3) in
rat neonatal myocytes. Our experiments involved experimental
hypoxia/ischaemia and we used only Fluo 3 measurements on a
MRC600 (laser scanning confocal) - under these conditions
rising cytosolic calcium could be tracked for up to 48 hours.
Obviously the may be SOME leakage of fluorophore, and I
suppose it would probably be good to measure Fluo 3 at
its isosbestic point on a second channel (CAN anyone comment
on the feasibility of this ?) to check for this.
I would be wary, however, of extended UV excitation (if you use
Indo or Fura) under confocal conditions. With the regular video
microscopy and ratio imaging, one also needs to excercise
discipline in viewing/grabbing images, and this is
progressively more important as you move to slit scanning
(KS2BIO) and laser scanning. It all depends on what you mean by
extended viewing I suppose, and what you do to
the cells, and which cells they are etc....
****************
> Even though it would be desirable to make free ccc
measurements with
> high temporal resolution (i.e., milliseconds), it would be satisfactory to
> be able to make free ccc measurements at several locations within a muscle
> at 10-15 min. intervals.
> Preparation will require use of a water or multi-immersion objective
> lens designed for use with or without a cover slip.
> 3-D imaging/reconstruction capability is not necessary.
> We are looking to spend $80,000 or less, preferably less than $70,000.
> We are considering a Nikon Optiphot-2 with the K2SBIO confocal
> attachment as well as a Zeiss Axioskop/Attofluor set-up (using deconvolution
> to achieve the desired confocal effect).
************
You may also want to check out the following disk scanning
systems:
1. Olympus confocal attachment [VX100] - can apparently be
attached to any microscope
2. Meridian's InsightPlus: Meridian also have software for
image acquisition, display and ratioing etc.
3. Bio-Rad's ViewScan DVC-250:
Note that all the above systems have single cameras as
detectors, rather than the 2 or 3 PMT detectors of laser
scanning systems. Your finances would seem, however, to dictate
a choice of a Nipkow disk or slit scanner system. I recommend
you try and get a demo of each of these systems using your
exact experimental conditions to check out each system. They
are all reasonably fast scanners geared up for phast physiology
experiments, but there may be additional expense in dual
imaging if you need filter changes, control software etc. Be
aware of that.
If you do get to try these systems, please post your
experiences to the group.
G'luck
Ian
*********** ************
Ian S Harper, PhD Int. Tel #: 27-21 938 0347
Experimental Biology Programme Int. Fax #: 27-21 938 0456
Medical Research Council Internet: [log in to unmask]
PO Box 19070 Tygerberg, 7505
South Africa
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