Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes Journal Article
| Authors: | Roorda, R. D.; Hohl, T. M.; Toledo-Crow, R.; Miesenboeck, G. |
| Article Title: | Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes |
| 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. |
| Keywords: | controlled study; unclassified drug; microscopy; nonhuman; animal cell; animals; microscopy, confocal; fluorescence; green fluorescent protein; ph; videorecording; luminescent proteins; cell membrane; green fluorescent proteins; contrast medium; drosophila melanogaster; microscopy, fluorescence, multiphoton; optics; photon; complementary dna; anisotropy; dipole; dna, complementary; xenopus; chromatophore; hippocampus; laser; neurotransmitter; olfactory receptor neurons; synapse vesicle; microscopy, video; nerve cell membrane; female; priority journal; article; synapto phluorin; optical probe |
| Journal Title: | Journal of Neurophysiology |
| Volume: | 92 |
| Issue: | 1 |
| ISSN: | 0022-3077 |
| Publisher: | American Physiological Society |
| Date Published: | 2004-07-01 |
| Start Page: | 609 |
| End Page: | 621 |
| Language: | English |
| DOI: | 10.1152/jn.00087.2004 |
| PROVIDER: | scopus |
| PUBMED: | 14999051 |
| DOI/URL: | |
| Notes: | J. Neurophysiol. -- Cited By (since 1996):54 -- Export Date: 16 June 2014 -- CODEN: JONEA -- Source: Scopus |
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25Toledo-Crow -
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