Electrochromic Voltage-Sensitive Dyes

Palette of fluorinated voltage sensitive dyes from Potentiometric Probes

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.

Help selecting filters is available and ElectroFluor products such as ElectroFluor560 include recommended Semrock filters

Stark Effect

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.

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