Spectral properties of the voltage-sensitive dye di‑4-ANBDQBS.

Abstract

Voltage-sensitive fluorescent dyes allow non-invasive recording of action potentials in excitable cells. The far-red-shifted voltage-sensitive dye, di-4-ANBDQBS (CytoVolt1), is stable for hours and produces optical-voltage changes through the Stark effect. However, the properties and precise voltage responses of the CytoVolt1 emission spectrum in living cells are still unclear. To better understand these changes, HEK293 cells were loaded with CytoVolt1, and emission scanning was performed using a confocal system at several excitation wavelengths. The average emission wavelength peak was 689+/- 2.3 (n = 21 cells;+/- SEM) at an excitation of 528 nm. In addition, peak emission wavelengths changed according to the excitation wavelength, with average peaks at 692+/- 3.3 nm and 705+/- 2.8 nm (n = 9, 9; p < 0.05) for 480 nm and 640 nm excitation wavelengths, respectively. We next wanted to know whether the voltage-dependence of fluorescence (as determined by the Stark shift) was linear at different excitation/emission wavelengths. We simultaneously voltage-clamped the CytoVolt1-stained HEK293 cells and measured fluorescence response at 720 nm (long-pass) for a physiological range of Vm values. With an excitation wavelength of 660 nm, the fluorescence-voltage relationship was linear (R2 = 0.995) with a slope of −0.0051 mV−1 (n = 6). Similarly, when the excitation wavelength was changed to 528 nm, the slopes for the fluorescence-voltage relations were 0.0013 mV−1 (R2= 0.996) and −0.0007 mV−1 (R2 = 0.998) for emissions at 650 nm and 769 nm (bandpass) (n = 10 cells), respectively. Dividing the two emission signals (650 nm/769nm) increased the slope to 0.0021 mV−1 and retained a high degree of linearity (R2 = 0.999). The data demonstrate that CytoVolt1 shows an emission shift corresponding to the excitation wavelength. CytoVolt1 also shows linear fluorescence-voltage relationships at different excitation and emission wavelengths, giving it utility as a voltage-sensitive fluorescent dye with different experimental applications.