Electrochromic Voltage-Sensitive Dyes
Voltage-Sensitive Dyes and the Stark Effect
Dyes that use the Stark effect are called electrochromic because there is a small but instantaneous shift in spectra in response to changes in membrane potential.
In order to convert this shift to large relative changes in fluorescence, measured as ∆F/F (%), careful excitation wavelength and filter choices must be made.
Stark Effect Figure Using ElectroFluor560 Spectral Data
- Absorption spectra of ElectroFluor560 (blue solid line) is shown along with left-shifted version (blue dashed line) as predicted for a Stark effect dye on a depolarized cell membrane
- Excitation wavelengths are selected to take advantage of changes in the absorption spectrum as shown in the figure. The emission spectrum also changes and can be used to generate a signal (not shown).
- Take home message: excite the dye at the “wings”, not the peak
- Exciting at the peak absorption does not produce a significant change in excitation or brightness and sensitivity is essentially zero.
- Exciting at the blue or red wings using wavelengths shorter or longer than the peak absorbance wavelength, respectively, yields voltage sensitivity.
- Exciting at the red edge is depicted in the inset box, where baseline fluorescence declines faster than the change in absorbance and ∆F/F can be large (close to 20%/100mV for some dyes) before absorbance is too inefficient to be practical.
- With dyes applied externally, the spectra shifts to the blue (left, as shown) during depolarization and red-edge excitation produces a decrease in fluorescence or inverted signal.
- Blue-edge excitation produces non-inverted signals for external, or bath-applied dyes, and signal polarities are inverted when dyes are applied internally.
- Voltage sensitivity using blue-edge excitation is weaker than when using red-edge excitation but is still very useful for ratiometric imaging where the ratio of blue-edge to red-edge signals (non-inverted/inverted) is taken.
- Dual-excitation wavelength voltage imaging can be implemented using a standard epifluorescence microscope with 2 rapidly switched LEDs exciting the dye at the blue and red wings of its spectrum in odd and even frames of a fast camera.
- Ratiometric imaging can remove artifacts due to motion, uneven staining, or bleaching and can also boost sensitivity.