Biomedical Engineering Reference
In-Depth Information
to monitor, at multiple sites, subthreshold events as they travel
from the sites of origin on neuronal processes and summate at
particular locations to influence action potential initiation. This
can be achieved by using voltage-imaging. Recently, the sensitiv-
ity of intracellular voltage-sensitive dye technique has been dra-
matically improved allowing direct recording of subthreshold and
action potential signals from the neurites of individual neurons
(10-12, 21-24)
.
We carried out experiments to develop a methodology for opti-
cal monitoring of the generation and spread of subthreshold
synaptic potentials and action potentials in neuronal processes
using selective staining of individual invertebrate and vertebrate
neurons by intracellular application of membrane impermeant
voltage-sensitive dyes
(25)
. First, we tested analogues of the most
successful absorption dyes known from extracellular application
because absorption dyes were substantially better in terms of pho-
todynamic damage as compared to fluorescence dyes. A total of
30 dyes were tested. The best results were obtained with two
positively-charged pyrazolone-oxonol dyes (designated JPW1177
and JPW1245), and two negatively-charged merocyanine dyes
(designated WW375 and JPW1124). However, the signal-to-
noise ratio for the best dyes in these series varied from 1 to 4
in single trials. We concluded that this mode was probably not
sensitive enough to allow monitoring neuronal signals from distal
processes.
Fluorescence measurements are more effective than absorp-
tion measurements in the situation where the image of the object
(e.g. thin process) is much smaller than the size of a photodetec-
tor
(6, 26)
. We found that JPW1114 was better than the three
other fluorescent styryl molecules we tested (RH461, RH437
and JPW1063) as determined by signal-to-noise ratio. There was
an improvement in sensitivity (as defined by the signal-to-noise
ratio) by a factor of about 50 over previously reported (e.g.
(27, 28)
) absorption and fluorescence signals from neuronal pro-
cesses stained by either intra- or extracellular application of dyes
(25)
. The obtained sensitivity depends on using fluorescence mea-
surements and selective staining of only one neuron within a gan-
glion
(29)
, and, on finding a dye that gives relatively large signals
when applied internally. The fluorescent voltage-sensitive styryl
dye JPW1114 (also called di-2-ANEPEQ), is a doubly positively
charged analog of the ANEPPS series of lipophilic voltage sen-
sitive dyes that is still sufficiently water soluble to be used for
microinjection. JPW1114 (available from Invitrogen - Molecular
Probes Inc. as D6923), together with its close analog JPW3028
(di-1-ANEPEQ), is presently the optimal voltage indicator for
intracellular application in recording from neuronal processes of
individual neurons in brain slices
(12, 23)
.
2.1.1. Dye Screening
