🚀 go-pugleaf

My apologies, List. There was no address for direct reply.

>
> Has anyone tried to visualise Aqueorine (a luminescent ~488nm calcium
> probe) using the PMTs on a confocal?
>
>
> Douglas Armstrong,
> University of Glasgow.
>

I have some experience with detection of aequorin and the MRC-600 but
don't want to hang out a shingle as an expert. As concisely as possible,
it all depends on what you are doing, but I'd eat my hat if you can image
single cells at the resting calcium level (approx. 100nM). This calcium
level approaches the Ca-independent emission level of aequorin, and you
would require an impossibly high concentration of aequorin in the cell.
The photomultiplier systems in the BIORAD instruments are not capable of
detecting single photon events, and the emission of the photons is not
going to be correlated to the scanning/sampling so even a local hotspot
would be smeared over the whole image array.

Since the photon emission of aequorin rises with about the 2.5th power of
[Ca], one might be able to detect a large cell being lysed into 1mM Ca
medium, but the event would be brief, don't blink. There are some
"improved" aequorins that have a higher output near the toe of the
emission curve, but I don't think they would help here.

In my former incarnation, I did quite a bit of single cell
intracellular calcium measurement work, and we only had good success when
FURA-2-DEXTRAN (the dextran is critical!!) became available.

I can send some refs if it's any interest to you. Please contact me directly.

Dale Callaham
Central Microscopy Facility
University of Massacusetts
Amherst, MA 01003

dac@bio.umass.edu