Second-harmonic generation voltage imaging at subcellular resolution in rat hippocampal slices

Action potential (AP) is a major signaling mechanism in the neuronal networks. Dendritic AP propagation is important for information processing within the individual neurons. Due to limitations of electrode-based techniques most research on subcellular AP propagation has been restricted to soma and proximal parts of the primary dendrites. Development of voltage-sensitive dyes (VSD) has opened up a possibility to measure voltage changes in the oblique dendrites and the spines. Membrane-bound organic VSD can be used both for fluorescent imaging and imaging of second-harmonic generation (SHG). Both phenomena are voltage dependent and can be used for measuring membrane potential changes. However, changes in SHG are linear to the change in the local membrane potential and its slope is constant across different compartments of cells. Although SHG demonstrates reasonable change with membrane voltage (over 10% per 100 mV), the signal-to-noise (S/N) ratio is currently lower in SHG measurement than in fluorescent methods.