Biomedical Engineering Reference
In-Depth Information
calcium imaging, the exposure of the preparation to the excita-
tion light was limited to the recording periods of 400 ms.
Even after 50-100 recording trials, typically collected for one
experiment, no change has been found in the amplitude or the
time course of optical or electrical signals due to photodynamic
damage (toxicity induced by the interaction of the indicator dye
with high intensity light).
The voltage-sensitive dyes, JPW 1114 or JPW 3028, have lit-
tle or no pharmacological effect when applied at functional con-
centrations to both invertebrate
(22, 25)
and vertebrate neurons
(11, 12, 21, 23, 24)
. We also demonstrated, for invertebrate neu-
rons, rat neocortical layer V and hippocampal CA1 pyramidal
neurons, and mitral cells of the olfactory bulb, that photodynamic
damage during optical recording was not present if the exposures
of the dendritic arbor to excitation light were kept relatively short
(100 ms) and if successive trials were separated by dark intervals
lasting several minutes
(10, 12, 21, 23, 24)
.
The current sensitivity of voltage imaging allowed experiments in
which subthreshold, synaptic potentials were monitored at the site
of origin, in the dendritic tuft of mitral cells in the olfactory bulb
of the rat.
Figure 3.4-I
illustrates presently available signal-to-
noise ratio obtained at the desired spatial resolution. Our results
showed consistently that the highest sensitivity was achieved in
recording from thin terminal branches in the mitral cell tuft, likely
due to the favorable surface-to-volume ratio of these structures.
The effect is based on the fact that, in addition to the plasma
membrane, the cytoplasm and the internal membranes (e.g. ER
membranes, mitochondria) of the stained cell also contain the
dye. Dye bound to internal membranes does not contribute to
the signal because it is not in the membrane that changes poten-
tial. This internal dye, nonetheless, contributes to the resting flu-
orescence and degrades the fractional change in light intensity
(
2.1.3. Example Results
F/F). In addition, the higher sensitivity of recording from the
tuft is accompanied by an improved signal-to-noise ratio (for the
same
F/F) based on a large membrane surface area of the termi-
nal dendritic arborization. Larger membrane surface area results
in the larger amount of the membrane bound dye and higher
light intensity; higher light intensity, in turn, results in the higher
signal-to-noise ratio under our experimental conditions (domi-
nant noise is statistical, shot noise). This is a fortunate feature
of voltage imaging because thin dendritic branches receive all of
the excitatory synaptic inputs in mitral cells, are not accessible to
direct electrical measurement, and are likely to be a key compart-
ment for signal integration.
Figure 3.4-I
illustrates the spatial resolution and the sensi-
tivity of voltage imaging from small dendritic structures (single
pixels received light from 4
×
4
μ
m areas in the object plane).
