>We are trying to locate a perfusion stage that will be compatable with
>the Z-stage of our Leica confocal.
>
>        Douglas W. Cromey, M.S.
>        Cell Biology and Anatomy
>        Arizona Health Sciences Center
>        1501 N. Campbell Ave.
>        Tucson, AZ  85724
>        (602)626-2824                           [log in to unmask]
>
>
We have spent some effort in developing a chamber for controlled temperature
perfusion on the stage of the Leica inverted. This has now been tested and
we are about to begin routine use with cultured cells grown on cover
glasses. The main features are:
 
1. Weight is around 220g (including the circulating water used for
temperature control). This is comfortably within the 250g limit specified by
the Leica factory engineers.
 
2. Thermal gradients (that cause out-gassing from perfusates and unstable
thermal profiles in the bath chamber) have been eliminated by water
jacketting of bath and lenses. Smooth flow ensures low vibration in the
water jackets. Inlet and outlet temperatures are monitored by thermocouples.
 
3. Two perfusate channels are provided with solution switching operated
hydraulically (no electrical noise). Switching occurs within the chamber
giving very small dead volume and rapid solution changes. A roller valve
ensures vibration-free switching so as not to disturb cells during optical
recording. Also, pressure changes that would distort the cover glass are
minimised. Perfusate flow is controlled by downstream resistance.
 
4. Chamber dimensions are 4.0mm x 200micrometres providing for rapid
perfusion of cells if required. This dimension indicates the area of cells
that can be observed by moving the stage and is limited by the dimension of
the outside of the objective lens.
 
5. A coverglass holder is press-fitted (by hand) into the bath allowing
various types of substrate to be accomodated.
 
6. All optical requirements of the Leica IRB and IRBE inverted 'scopes are
catered for e.g. working distances of condensers (23mm), objective
clearances etc.
 
7. We are now wrestling with the problem of incorporating micromanipulators
into the bath so as to do patch-clamp and microelectrode work.
 
 
If this is of interest to you or to anyone else, we could follow it up with
Leica to see if they will commission manufacture of a bunch of them and
distribute them as accessories. If there is any interest out there, please
let me know.
 
 
Peter Harris
Department of Physiology
The University of Melbourne,
Parkville
Victoria 3052
AUSTRALIA
 
Fax.  61 3 344 5818
Tel.  61 3 344 5841