Biology Reference
In-Depth Information
Abstract
This chapter compares the imaging capabilities of a range of systems including
multiphoton microscopy in regard to measurements of intracellular Ca
2
þ
within
living cells. In particular, the excitation spectra of popular fluorescent Ca
2
þ
indicators are shown during 1P and 2P excitation. The strengths and limitations
of the current indicators are discussed along with error analysis which highlights
the value of matching the Ca
2
þ
a
nity of the dye to a particular aspect of Ca
2
þ
signaling. Finally, the combined emission spectra of Ca
2
þ
and voltage sensitive
dyes are compared to allow the choice of the optimum combination to allow
simultaneous intracellular Ca
2
þ
and membrane voltage measurement.
Y
I. Why Study Ca
2
þ
Signaling with Confocal and Multiphoton
Microscopy
Ca
2
þ
is a ubiquitous intracellular messenger that controls a large number of
cellular processes, such as gene transcription, excitation, contraction, apoptosis,
cellular respiration, and the activity levels of many cell-signaling messenger cas-
cades. Inside the cell, Ca
2
þ
may, under various conditions, sequester into the sarco/
endoplasmic reticulum, mitochondria, and the nucleus, or exist in the cytosol
either in its free form or as bound to bu
ers. Typically, a large Ca
2
þ
concentration
gradient is maintained across the plasma membrane of the cell. Because of di
V
erent
Ca
2
þ
channels, pumps, and exchangers on the membranes of the cell or organelles,
Ca
2
þ
fluxes may be created at multiple locations in the cell. Therefore, Ca
2
þ
concentration and signal may be specific with respect to both location and time.
Moreover, Ca
2
þ
may concentrate in distinct cytoplasmic regions because of tight
physical loci not enclosed by membranes, for example, the dyadic area between the
transverse tubule and the sarco/endoplasmic reticulum of muscle cells. Given the
large number of Ca
2
þ
channels feeding it with Ca
2
þ
from both the extracellular
space (transverse tubule) and the sarco/endoplasmic reticulum, such that the dyad
may transiently have very di
V
erent localized Ca
2
þ
concentrations compared to the
rest of the cytosol which may be only nanometers away. Thus, Ca
2
þ
localizes in the
cytosol as well as within organellar compartments, and these Ca
2
þ
signals may last
for very short timeframes (ns) or for substantially longer periods of time (min).
Ca
2
þ
signaling per se is not within the remit of this chapter and will not be covered
in any detail, but the interested reader is referred to other sources, for example,
Bootman et al. (2001)
.
Nonetheless, for the purpose of this chapter, it is important to acknowledge that
the average Ca
2
þ
concentration in any given cell usually ranges 0.01-1
m
M, but
that the Ca
2
þ
almost never exists uniformly across the cell, and that local Ca
2
þ
events may occur with very fast time courses. This therefore requires Ca
2
þ
imaging
of live specimens with high spatial and temporal resolution. Thus, one would
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