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.
Laboratory of Neural Systems, Memorial-Sloan-Kettering Cancer Center, Box 205, 1275 York Ave., New York, NY 10021, USA.
Robert D
RD
Roorda
Tobias M
TM
Hohl
Ricardo
R
Toledo-Crow
Gero
G
Miesenböck
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D018076:DNA, Complementary / Q000032:analysis* / Q000502:physiology; D004331:Drosophila melanogaster; D005260:Female; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods; D018034:Olfactory Receptor Neurons / Q000737:chemistry* / Q000502:physiology; D055095:Optics and Photonics; D014981:Xenopus
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins