How It Works

Dyes such as Di-4-ANEPPS  and Di-4-AN(F)EPPTEA  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 (Δ​F/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.