Biology Reference
In-Depth Information
These errors can be compounded by errors in estimates of fluorescence or the
variability of signals from one cell to the next.
Fig. 10
B shows the errors in Ca
2
þ
based on simple errors in fluorescence changes. The graph illustrates the risk
inherent in using dyes with a relatively high a
Y
nity relative to the physiological
signal. Small errors in the range of fluorescence signals translate to large errors of
intracellular Ca
2
þ
such that the ability to discriminate changes in maximum
physiological response is severely impaired. This can be significantly improved
by using lower a
nity dyes, but at the cost of poor resolution of minimum or
background intracellular [Ca
2
þ
].
Y
XX. Multimodal and Multiple Fluorophore Confocal and
Multiphoton Microscopy
Although Ca
2
þ
is an important signaling molecule in a variety of cell types, it by
no means operates alone. Rather, Ca
2
þ
both temporally and spatially interacts
with many other properties and processes in the cell that only in concert orches-
trate cellular function. Thus, some of these processes are dictated by Ca
2
þ
, but
some or not. A good example of this interplay is excitation-contraction coupling
in muscle cells, in which the action potential depolarizes the plasma membrane of
the cell, which causes a small influx of Ca
2
þ
through the membrane. This inward
Ca
2
þ
current stimulates the ryanodine receptor to release bulk Ca
2
þ
from the
sarcoplasmic reticulum, which upon binding to the myofilaments induces the
actin-myosin interaction and the subsequent cellular contraction (
Bers, 2002
).
The cellular contraction may be imaged by simple black-and-white contrast
edge-detection microscopy, but this is not the case for the intracellular Ca
2
þ
and
membrane potential characteristics that both require more sophisticated methods
such as fluorescence microscopy. Thus, simultaneous imaging with the use of
multiple fluorophores present at the same time in the specimen or combinations
of di
erent imaging modalities in some sense is required for capturing complex
information.
Thus, loading or injecting the specimen with multiple fluorophores allows for
simultaneous recording of di
V
V
erent signals, or if simultaneous recordings are not
technically possible, di
erent signals may be recorded sequentially without having
to manipulate, move, or in any other way perturb the specimen between recordings.
In the latter case, only the optical pathways of the microscope would be altered
between recordings, whereas the specimen would not, since it would already be
loaded with di
V
erent fluorophores. The use of multiple fluorophores require either
the ability to direct separate emission wavelength bands onto di
V
V
erent light detec-
tors, or to spectrally separate di
erent excitation wave-
lengths. Depending on the hardware, both confocal and multiphoton microscopes
can fulfill these requirements and therefore allow for measurements with multiple
fluorophores. Such experiments can be done by simultaneously loading the
V
erent fluorophores by di
V
