Voltage-Sensitive Dyes (VSDs)
How VSDs Work
Dyes such as Di-4-ANEPPS and Di-4-AN(F)EPPTEA (ElectroFluor530TM) fluoresce when bound to cell membranes. These fast-response probes change their fluorescence properties in response to changes in the surrounding electric field allowing an optical readout of transmembrane potential. The response is fast enough to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells, even intact brains and hearts (see citations). Although the magnitude of their potential-dependent fluorescence change is often small (2–10% fluorescence change per 100 mV), excellent signal-to-noise can still be obtained thanks to bright and highly stable fluorescence. Fluorination was introduced as a way to improve photostability, which allows stronger excitation and less photobleaching. At the same time, fluorination allows spectra to be fine tuned, providing flexibility for integration with other fluorescent probes/labels.
Stark-shift electrochromic dye molecules are comprised of 3 main components: the fluorophore, polar head group, and membrane binding motif. Several variations of dyes based on a common fluorphore backbone are made by changing the polar head group, and lipophilic membrane binding domain. This is can be thought of as a toolkit to modify solubility/membrane binding properties, fine tune spectral properties, etc. Fluorination is a final modification that improves photostability and shifts spectra producing a “palette” of voltage-sensitive dyes as shown above.
While general information about dye properties can be found here, contact Potentiometric Probes for recommendations of the best dye for your specific application and imaging modality.
Choosing the best wavelength for your measurement
Most of our VSDs (with the exception of TMRE and TMRM) work via an electrochromic (aka Stark effect) mechanism, whereby the spectrum shifts in response to a change in membrane potential. Therefore, the best wavelengths for voltage sensitivity are at the wings of the spectrum rather than at the peak. We generally recommend excitation at the red edge because the red wing of the spectrum is generally steeper than the blue wing. In general, there needs to be a compromise between high fractional voltage sensitivity (ΔF/F), achieved by red-edge excitation where the total fluorescence is low, and optimal signal to noise, achieved by excitation closer to the peak of the spectrum. Dual wavelength ratio measurements (red edge/blue edge) can also be implemented to improve the sensitivity and to normalize away uneven staining or motion artifacts.
The two main backbones
ANEP: (AminoNaphthylEthenylPyridinium) with a peak absorption wavelength range from 450 to 510nm.
ANBDQ: (AminoNapthylButydienylQuinolinium) red shifted to longer wavelengths, peak absorption wavelength range from 530 to >550nm.
Membrane binding motifs
Di-4/Di-2: Describes the dye’s two membrane binding alkyl/hydrocarbon chains with 4 or 2 carbons for example. Generally speaking a longer hydrocarbon tail improves membrane binding and reduces aqueous solubility.
Dye solubility depends strongly on several factors. Dyes with a propyl-sulfonate (PS) polar head group and butyl chains such as Di-4-ANEPPS are not very soluble in aqueous solutions. VSDs with TEA (triethylamine) head group and ethyl chains such as Di-2-AN(F)EPPTEA (ElectroFluor530sTM) are highly soluble in aqueous solutions. Lower solubility leads to longer staining persistence. Higher solubility improves tissue penetration and is useful for intracellular application through a patch pipet.
These patented VSDs deliver enhanced sensitivity and photostability by strategic placement of fluorine substituents in the chromophore. An (F) appears in the dye’s name at the location matching the fluorine’s location. For example Di-4-AN(F)EPPTEA (ElectroFluor530TM) is the same as Di-4-ANEPPTEA except with a fluorine on the naphthyl. The location-dependent spectral shift results in a “palette” of VSD variants, where the numerical suffix in ElectroFluor(Wavelength) indicates the excitation wavelength with optimal voltage sensitivity.
Dye Packaging and Storage
Dyes are sold as pre-aliquoted, pre-dried amounts suitable for typical assays for convenience. The tubes of dried dye can be stored easily (<4°C with desiccant, in the dark) saving the user extra steps of preparing stock solutions and removing solvents.