Biomedical Engineering Reference
In-Depth Information
the membrane whose potential is changing
(26, 82, 83)
. Gonzalez
and Tsien
(84)
introduced a new scheme for generating voltage-
sensitive signals using two chromophores and fluorescence res-
onance energy transfer (FRET). While these fractional changes
were also in the range of 10%/100 mv, more recent results are
about 30% (Gonzalez and Tsien, personal communication). How-
ever, in order to achieve fast response times (
100 Hz), one of
the chromophores must be very hydrophobic and, as a result,
does not penetrate into brain tissue. Thus far, it has not been pos-
sible to measure fast signals with a fast pair of dyes in intact tissues
(Gonzalez and Tsien; Obaid and Salzberg; personal communica-
tion), although impressive results have been obtained where speed
was not critical
(85, 86)
.
Membrane potential changes the non-linear second harmonic
generation from styryl dyes
(87-89)
. Large (50%) fractional
changes were measured. But, because the number of detected
photons is small, the signal-to-noise ratios remain substantially
smaller than that obtained with wide-field measurements.
>
5.3. Neuron-Type
Specific Staining
An important new direction is the development of methods for
neuron-type specific staining which would make it possible to
determine the role of specific neuron types in generating the
input-output function of a brain region. Three quite different
approaches have been tried.
The use of anterograde and retrograde dye transport has
resulted in specific staining of olfactory receptor neuron terminals
(Friedrich and Korsching
(53)
; Example 3) and motor neurons in
embryonic chick and in lamprey spinal cords
(51)
. This method
depends on finding a location where only one cell type is present
that has a process leading to the brain area of interest. In lamprey
experiments, spike signals from individual neurons were some-
times measured
(90)
. Further effort at optimizing this staining
procedure is needed.
5.3.1. Directed Transport
The use of cell-type specific staining developed for fluorescein by
Nirenberg and Cepko
(91)
might be extended to ion-sensitive or
voltage-sensitive dyes.
5.3.2. Staining
Dependent on
β-galactosidase
Expression
Siegel and Isacoff
(92)
constructed a genetically encoded com-
bination of a potassium channel and green fluorescent protein.
When expressed in a frog-oocyte, this molecule, FlaSh, had a (rel-
atively slow; few hundred millisecond) voltage-dependent signal
with a fractional fluorescence change of 5%. More recently, Ataka
5.3.3. Genetically
Encoded Activity Sensors
5.3.3.1. Voltage Sensors
