Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes.
Academic Article
Overview
abstract
Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.