Above: extitation and emission of the voltage sensitive dye, main components of the experimental setup. Below: The ability of Di-4-ANEPPS (one of the Voltage - Sensitive Dye) to bind into the cell membrane. The dye binds perpendicular to the membrane, which turns out to be parallel to the electric field incurred during an action potential (Source: Peter Baker, Dep. of Biology, Arizona State University).
Top: the chemical structure of a voltage-sensitive dye RH 795. The 4 test tubes below the dye structure contain another voltage-sensitive dye dissolved in 4 different solvents of very different polarity. The large changes in its color indicate that this dye is sensitive to its microenvironment.
Bottom: schematic illustration of the dye interactions with the lipid bi-layer, depicting one out of many possible mechanisms responsible for a dye ability to transduce the membrane potential change into an optical signal. Dye molecules not bound to the lipid bi layer are not fluorescent (depicted in blue). Once the dye binds it becomes fluorescent (red). The intensity of the fluorescence depends on the extent that the dye hydrophobic portion interacts with the hydrophobic portion of the bi-layer. Since the dye is both charged and polar, it may change its interaction with the bi-layer, depending on the electric field across the
bi-layer. An action potential gives rise to a change in electric field
of ~ 20,000 Volts across the bi-layer. Such a large electric field
change can also induce an electro-chromic effect even if the dye does not move during an action potential
Calcium Green Mechanism :
Upon binding with calcium, the aminodiacetate group of Calcium Green is less likely to transfer a fluorescence-quenching electron to the fluorophore,
allowing the fluorophore to emit a photon. The peak absorption
wavelength for this molecule is 506nm and maximum emission
occurs at 531nm (Yuste et al. 2000 ; Molecular Probes, 2001).
Examples of the In Vivo Voltage-Sensitive Dye Optical Imaging
1.Somatosensory (Vibrissae representation at the Barrel Field)
2.Auditory (tonotopicity)
Examples of the In Vivo Voltage-Sensitive Dye Optical Imaging
1.Somatosensory (Vibrissae representation at the Barrel Field)
2.Auditory (tonotopicity)
3.Visual (orientation column)
Main feautres of the Voltage-Sensitive Dye:
Main feautres of the Voltage-Sensitive Dye:
Temporal resolution:
-From low to ~0.1 ms
Spatial resolution:
- In depends of ROI, up to 1 mcm
-Toxicity:
From moderate to high
-lifetime: from 10 min to few hr
-Application: In Vivo and In Vitro but not in human research
-Main advantage:
Answer for question not only “where?” abut also “when?”