Voltage-sensitive dyes, also known as potentiometric dyes, are

dyes A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...

which change their

spectral ''Spectral'' is a 2016 3D military science fiction, supernatural horror fantasy and action-adventure thriller war film directed by Nic Mathieu. Written by himself, Ian Fried, and George Nolfi from a story by Fried and Mathieu. The film stars J ...

properties in response to

voltage Voltage, also known as electric pressure, electric tension, or (electric) potential difference, is the difference in electric potential between two points. In a static electric field, it corresponds to the work needed per unit of charge to ...

changes. They are able to provide linear measurements of firing activity of single

neurons A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa ...

, large neuronal populations or activity of myocytes. Many physiological processes are accompanied by changes in cell

membrane potential Membrane potential (also transmembrane potential or membrane voltage) is the difference in electric potential between the interior and the exterior of a biological cell. That is, there is a difference in the energy required for electric charges ...

which can be detected with voltage sensitive dyes. Measurements may indicate the site of

action potential An action potential occurs when the membrane potential of a specific cell location rapidly rises and falls. This depolarization then causes adjacent locations to similarly depolarize. Action potentials occur in several types of animal cells ...

origin, and measurements of action potential velocity and direction may be obtained. Potentiometric dyes are used to monitor the electrical activity inside cell organelles where it is not possible to insert an

electrode An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit (e.g. a semiconductor, an electrolyte, a vacuum or air). Electrodes are essential parts of batteries that can consist of a variety of materials ...

, such as the

mitochondria A mitochondrion (; ) is an organelle found in the cells of most Eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used ...

and dendritic spine. This technology is especially powerful for the study of patterns of activity in complex multicellular preparations. It also makes possible the measurement of spatial and temporal variations in

along the surface of single cells.

Types of dyes

Fast-response probes: These are amphiphilic membrane staining dyes which usually have a pair of hydrocarbon chains acting as membrane anchors and a hydrophilic group which aligns the

chromophore A chromophore is the part of a molecule responsible for its color. The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the mo ...

perpendicular to the membrane/aqueous interface. The chromophore is believed to undergo a large electronic charge shift as a result of excitation from the ground to the

excited state In quantum mechanics, an excited state of a system (such as an atom, molecule or nucleus) is any quantum state of the system that has a higher energy than the ground state (that is, more energy than the absolute minimum). Excitation refers to ...

and this underlies the putative electrochromic mechanism for the sensitivity of these dyes to membrane potential. This molecule (dye) intercalates among the

lipophilic Lipophilicity (from Greek λίπος "fat" and φίλος "friendly"), refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. Such non-polar solvents are themselves lipo ...

part of

biological membrane A biological membrane, biomembrane or cell membrane is a selectively permeable membrane that separates the interior of a cell from the external environment or creates intracellular compartments by serving as a boundary between one part of the ...

s. This orientation assures that the excitation induced charge redistribution will occur parallel to the

electric field An electric field (sometimes E-field) is the physical field that surrounds electrically charged particles and exerts force on all other charged particles in the field, either attracting or repelling them. It also refers to the physical field ...

within the membrane. A change in the voltage across the membrane will therefore cause a spectral shift resulting from a direct interaction between the field and the ground and excited state dipole moments. New voltage dyes can sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. Slow-response probes: These exhibit potential-dependent changes in their transmembrane distribution which are accompanied by a fluorescence change. Typical slow-response probes include cationic carbocyanines and rhodamines, and ionic oxonols.

Examples

Commonly used voltage sensitive dyes are substituted aminonaphthylethenylpyridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and RH237. Depending on their chemical modifications which change their

physical properties A physical property is any property that is measurable, whose value describes a state of a physical system. The changes in the physical properties of a system can be used to describe its changes between momentary states. Physical properties are ...

they are used for different experimental procedures. They were first described in 1985 by the research group of Leslie Loew.

ANNINE-6plus ANNINE-6plus is a water soluble voltage sensitive dye (also called potentiometric dyes). This compound was developed at the Max Planck Institute for Biochemistry in Germany. It is used to optically measure the changes in transmembrane voltage of ...

is a voltage sensitive dye with fast response (ns response time) and high sensitivity. It has been applied to measure the action potentials of a single t-tubule of cardiomyocytes by Guixue Bu et al. More recently, a series of fluorinated ANEP dyes was introduced that offer enhanced sensitivity and photostability; they are also available over a wide choice of excitation and emission wavelengths. A recent computational study confirmed that the ANEP dyes are affected only by the electrostatic environment and not by specific molecular interactions. Other structural scaffolds, such as xanthenes, are also successfully used.

Materials

The core material for imaging brain activity with voltage-sensitive dyes are the dyes themselves. These voltage-sensitive dyes are

and preferably localized in membranes with their hydrophobic tails. They are used in applications involving fluorescence or absorption; they are fast acting and are able to provide linear measurements of changes in membrane potential. Voltage sensitive dyes are supplied by many companies who offer

fluorescent probes A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...

for biological applications
Potentiometric Probes, LLC
specializes only in voltage sensitive dyes; they have an exclusive license to distribute the large set of fluorinated VSDs, marketed under the ElectroFluor^TM brand. A variety of specialized equipment may be used in conjunction with the dyes, and choices in equipment will vary according to the particularities of a preparation. Essentially, equipment will include specialized microscopes and imaging devices, and may include technical lamps or lasers.

Strengths and weaknesses

Strengths of imaging brain activity with voltage-sensitive dyes include the following abilities: * Measurement of population signals from many areas may be taken simultaneously, and hundreds of neurons may be recorded from. Such multisite recordings may provide precise information on action potential initiation and propagation (including direction and velocity), and on the entire branching structure of a neuron. * Measurements of spike activity in a ganglion that is producing behaviour can be taken and may provide information about how the behaviour is producing. * In certain preparations the pharmacological effects of the dyes may be completely reversed by removing the staining pipette and allowing the neuron 1–2 hours for recovery. * Dyes may be used to analyze signal integration in terminal dendritic branches. Voltage-sensitive dyes offer the only alternative to genetically encoded

voltage sensitive proteins Voltage, also known as electric pressure, electric tension, or (electric) potential difference, is the difference in electric potential between two points. In a static electric field, it corresponds to the work needed per unit of charge to ...

(such as Ci-VSP derived proteins) for doing this. * More soluble dyes such as
ElectroFluor-530s
o
di-2-ANEPEQ
may perfused internally into single cell through a patch pipet. This technique has permitted the study of electrical signals in individual dendrites and dendritic spines within brain slices. Weaknesses of imaging brain activity with voltage-sensitive dyes include the following problems: * Voltage-sensitive dyes may respond very differently from one preparation to another; typically tens of dyes must be tested in order to obtain an optimal signal., imaging parameters, such as excitation wavelength, emission wavelength, exposure time, should also be optimized. * Voltage-sensitive dyes often fail to penetrate through

connective tissue Connective tissue is one of the four primary types of animal tissue, along with epithelial tissue, muscle tissue, and nervous tissue. It develops from the mesenchyme derived from the mesoderm the middle embryonic germ layer. Connective tiss ...

or move through intracellular spaces to the region of membrane desired for study. Staining is a serious issue in applications of these dyes. Water-soluble dyes, such as

ElectroFluor-530s
o
di-2-ANEPEQ
do not suffer this problem. * On the other hand, if the dyes are too water-soluble, staining may not persist. This can be addressed by utilizing dyes containing longer alkyl chains to increase lipophilicity. * Noise is a problem in all preparations with voltage-sensitive dyes and in certain preparations the signal may be significantly obscured. Signal to noise ratios can be improved with

spatial filter A spatial filter is an optical device which uses the principles of Fourier optics to alter the structure of a beam of light or other electromagnetic radiation, typically coherent laser light. Spatial filtering is commonly used to "clean up" the ou ...

ing or temporal filtering algorithms. Many such algorithms exist; one signal processing algorithm can be found in recent work with the ANNINE-6plus dye. * Cells may be permanently affected by treatments. Lasting pharmacological effects are possible, and the photodynamics of the dyes can be damaging. Recently developed fuorinated voltages sensitive dyes have been shown to mitigate these effects.

Uses

Voltage-sensitive dyes have been used to measure neural activity in several areas of the nervous system in a variety of organisms, including the

squid giant axon The squid giant axon is the very large (up to 1.5 mm in diameter; typically around 0.5 mm) axon that controls part of the water jet propulsion system in squid. It was first described by L. W. Williams in 1909, but this discovery was f ...

, whisker barrels of the rat somatosensory cortex,

olfactory bulb The olfactory bulb (Latin: ''bulbus olfactorius'') is a neural structure of the vertebrate forebrain involved in olfaction, the sense of smell. It sends olfactory information to be further processed in the amygdala, the orbitofrontal cortex ...

of the salamander,

visual cortex The visual cortex of the brain is the area of the cerebral cortex that processes visual information. It is located in the occipital lobe. Sensory input originating from the eyes travels through the lateral geniculate nucleus in the thalamus ...

of the cat, optic tectum of the frog, and the visual cortex of the

rhesus monkey The rhesus macaque (''Macaca mulatta''), colloquially rhesus monkey, is a species of Old World monkey. There are between six and nine recognised subspecies that are split between two groups, the Chinese-derived and the Indian-derived. Generally ...

. Many applications in cardiac electrophysiology have been published, including ''ex vivo'' mapping of electrical activity in whole hearts from various animal species, subcellular imaging from single cardiomyocytes, and even mapping both sinus rhythms and arrhytmias in open heart ''in vivo'' pig, where motion artifacts could be eliminated by dual wavelength ratio imaging of the voltage sensitive dye fluorescence.

Types of dyes

Examples

Materials

Strengths and weaknesses

Uses

References

Further reading