MPE imaging of intracellular calcium ion activity
Our MPE
imaging of intracellular calcium ion activity (for
example see Piston et al., 1994) focused primarily
on local
release events or "sparks" in developing rat skeletal
muscle
(Zipfel et al., 1996). These studies were done in collaboration
with J.P. O'Malley
and M.M. Salpeter in Neurobiology and Behavior
at Cornell. Absolute calcium concentrations are calibrated using
Indo-1, a UV excited and ratiometric calcium indicator dye. Fluo-3
is used for dynamical measurements due to its superior signal-to-noise
properties. The figure below shows
a line scan "image" (the horizontal axis represents
time) of a Fluo-3 loaded myotube. For acquisition of each frame
a single
line is repeatedly scanned in the same location. The ~5µm
bursts of fluorescence are due to non-random calcium fluctuations
equivalent
to ~105-106 ions. The image at right shows
a ryanodine-labeled cell (blue) overlaid with sequentially ratioed
Fluo-3 images (red) that indicate only changes in ion activity.
Sparking events are located at particular recurring sites with
a preference
for those near specific nuclei.
D.W. Piston, M.S. Kirby, H. Cheng, W.J. Lederer and W.W. Webb. Applied Optics, 33:662, 1994.
W.R. Zipfel, J.P. O'Malley, D. Van Helden, R.M. Williams, J.B. Guild, M.M. Salpeter and W.W. Webb. Biophys. J., 70:A427, 1996.
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