>Hello,
>
>     I have two questions today.  1.  Does anyone have any opinions
>concerning Meridian InSIGHT?  2.  What do you think about the possibility of
>doing calcium measurements using the ratio of Fluo-3 to Fura-Red?
>___________________________________________________________________
>
>Dan Boorstein
>Muscle Biology Laboratory                      [log in to unmask]
>Texas A&M University                            [log in to unmask]
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Dear Dan
I have done fluo-3/fura-red calcium measurements using the Meridian.
Fluo-3/fura-red worked much better than I expected considering theoretical
problems of differing compartmentalization of dyes, differing efflux rates
and deesterification rates. What is a problem is balancing the 2 dyes to get
sufficient signal at each wavelength. With dual wavelength dyes this is not
a problem. So you need preliminary experiments to determine dye
concentrations and loading times and then it is reproducible for a
particular cell type.
 
As for the Meridian, I new to confocal but I had no complaints about the
video rate image capture. Software was OK but one problem we encountered was
with cells that moved during the time course (contracting endothelial
cells). The 'cookie cutter' maps around each cell couldn't be adjusted on
each time frame. I believe that Meridian is addressing this and overall
Meridian have been very responsive to such problems.
 
Another problem we had was the coverslip perfusion bath (Biophysica) for
maintaining the temperature. We had the water bath at about 45oC to
compensate for cooling in the tubes leading to the perfusion chamber. We
were using a fast flow to deliver agonist (approx 10s to change chamber
volume) This gave an increase in temperature in chamber which was sufficient
to cause coverslip to flex or something similar and confocal image dropped
out of focus momentarily. Actually an argument for not using confocal
although we now realize we get a similar effect on our Zeiss microphotometry
setup using a normal microscope that we hadn't noticed before. So the design
of perfusion bath is critical. Hope this helps
Gary Jamieson
Haematology
Austin Hospital
Heidelberg VIC 3084
AUSTRALIA
Internet [log in to unmask]
Phone    613 496 5518
Fax      613 459 1674