Image processing for non-ratiometric measurement of membrane voltage using fluorescent reporters and pulsed laser illumination

• An image processing technique for assessment of ANNINE-6 fluorescence is proposed.
• Fluctuations due to illumination by nanosecond dye laser are quantified.
• Fluctuations are suppressed using optical noise as correcting factor.
• Contribution of ASE is quantified.

The measurement of transmembrane voltages induced by pulsed electric field exposure can be achieved by using fluorescent dyes like ANNINE-6. Such approach requires a quantitative determination of the fluorescence intensity along the cell's membrane by image processing. When high temporal resolution is required, the illumination source is frequently a dye-laser which causes high fluctuations in the intensity of illumination which in turn affects the fluorescence intensity and thus the quality of the results. We propose an image processing technique that allows to overcome the fluctuations and to produce quantitative data. It uses the optical background noise as a correcting factor. Standard deviation in the fluctuations is thus efficiently reduced by at least a factor of 2.5. Additionally we draw attention to the fact that the parasitic component of the laser radiation (ASE) can also suppress fluctuations although it deteriorates wavelength precision.