Abstract
The feasibility of determining localized Ca$^2+$ influx using
only wide-field fluorescence images was explored by imaging (using
fluo-3) single channel Ca$^2+$ fluorescence transients (SCCaFTs),
due to Ca$^2+$ entry through single openings of Ca$^2+$-permeable
ion channels, while recording unitary channel currents. Since the
image obtained with wide-field optics is an integration of both in-focus
and out-of-focus light, the total fluorescence increase (DeltaF(total)
or "signal mass") associated with a SCCaFT can be measured directly
from the image by adding together the fluorescence increase due to
Ca$^2+$ influx in all of the pixels. The assumptions necessary
for obtaining the signal mass from confocal linescan images are not
required. Two- and three-dimensional imaging was used to show that
DeltaF(total) is essentially independent of the position of the channel
with respect to the focal plane of the microscope. The relationship
between Ca$^2+$ influx and DeltaF(total) was obtained using SCCaFTs
from plasma membrane caffeine-activated cation channels when Ca$^2+$
was the only charge carrier of the inward current. This relationship
was found to be linear, with the value of the slope (or converting
factor) affected by the particular imaging system set-up, the experimental
conditions, and the properties of the fluorescent indicator, including
its binding capacity with respect to other cellular buffers. The
converting factor was used to estimate the Ca$^2+$ current passing
through caffeine-activated channels in near physiological saline
and to estimate the endogenous buffer binding capacity. In addition,
it allowed a more accurate estimate of the Ca$^2+$ current underlying
Ca$^2+$ sparks resulting from Ca$^2+$ release from intracellular
stores via ryanodine receptors in the same preparation.
- 15337821
- aniline
- animals,
- bufo
- caffeine,
- calcium
- calcium,
- channel
- channels,
- compounds,
- conductivity,
- dyes,
- electric
- fluorescence,
- fluorescent
- gating,
- gov't,
- in
- ion
- marinus,
- microscopy,
- muscle,
- myocytes,
- p.h.s.,
- patch-clamp
- research
- smooth
- support,
- techniques,
- u.s.
- vitro,
- xanthenes,
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