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Date: | Sat, 16 Jun 2007 12:38:50 -0400 |
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We have been working on this off and on for over a year, slowly improving our sensitivity, but still
not good enough for real time application. We are currently using an Olympus IX-71 with a
Cascade II camera. With 100 ms exposures we can detect a spike followed by a decay when ATP is
added to the medium by micropipette. The current detection limit is around 1 micromolar final
concentration, but I am not sure whether the spike represents unmixed ATP that then equilibrates
or truly 1 µM that is then rapidly consumed. Given the concentrations of everything, it is probably
the former. In any case, we still cannot see the release from single cells at this acquisition rate,
which is what we want. Next step is to compare lenses for transmission and overall brightness.
There are several reports in the literature, so we think it is doable. All the papers I have seen use a
20x lens and a Princeton Instruments camera (back-thinned chip). Seems like the Cascade or other
EM camera should be competitve. From what I understand, it may come down to what is in the
light path between the specimen and the camera. In the meantime, a colleague has built a
macroscope with a Cascade II in a box that can see release from clusters of our cells with 200 ms
exposures. If you are interested in picking his brain, email me and I will forward your email to him.
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