Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes.

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

Characterization and fabrication of fully metal-coated scanning near-field optical microscopy SiO2 tips

The fabrication of silicon cantilever-based scanning near-field optical microscope probes with fully aluminium-coated quartz tips was optimized to increase production yield. Different cantilever designs for dynamic- and contact-mode force feedback were implemented. Light transmission through the tips was investigated experimentally in terms of the metal coating and the tip cone-angle. We found that transmittance varies with the skin depth of the metal coating and is inverse to the cone angle, meaning that slender tips showed higher transmission. Near-field optical images of individual fluorescing molecules showed a resolution < 100 nm. Scanning electron microscopy images of tips before and after scanning near-field optical microscope imaging, and transmission electron microscopy analysis of tips before and after illumination, together with measurements performed with a miniaturized thermocouple showed no evidence of mechanical defect or orifice formation by thermal effects