Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy

Although nonlinear microscopy and fast (∼1 ms) membrane potential (V_m) recording have proven valuable for neuroscience applications, their potentially powerful combination has not yet been shown for studies of V_m activity deep in intact tissue. We show that laser illumination of neurons in acute rat brain slices intracellularly filled with FM4-64 dye generates an intense second-harmonic generation (SHG) signal from somatic and dendritic plasma membranes with high contrast >125 μm below the slice surface. The SHG signal provides a linear response to ΔV_m of ∼7.5%/100 mV. By averaging repeated line scans (∼50), we show the ability to record action potentials (APs) optically with a signal-to-noise ratio (S/N) of ∼7-8. We also show recording of fast V_m steps from the dendritic arbor at depths inaccessible with previous methods. The high membrane contrast and linear response of SHG to AV_m provides the advantage that signal changes are not degraded by background and can be directly quantified in terms of ΔV_m. Experimental comparison of SHG and two-photon fluorescence V_m recording with the best known probes for each showed that the SHG technique is superior for V_m recording in brain slice applications, with FM4-64 as the best tested SHG V_m probe.