Line excitation array detection microscopy

Date

2019-07-16

Authors

Martin, Christopher Michael

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Abstract

High-speed, volumetric fluorescence and two-photon imaging systems are a necessity for studying biological systems. To screen drug compounds using small animal models, hundreds of animals must be imaged in seconds. To discover how the brain gives rise to behavior and disorders, neural circuits must be monitored at the millisecond timescales of action potentials. This dissertation develops a high-speed scanned light-sheet imaging method, called line excitation array detection (LEAD) microscopy, to meet the needs of such applications. LEAD microscopy combines fast line-scanning with an acousto-optic deflector and fast, sensitive imaging with a linear photomultiplier tube array to reach high frame rates. LEAD microscopy was first implemented as a high-throughput fluorescence cytometer for the small nematode Caenorhabditis elegans. An acousto-optic deflector scans an excitation line across a field-of-view covering the C. elegans cross-section at 800,000 Hz. Fourteen elements of a linear photomultiplier tube array image the line as it scans to generate 66×14 pixel frames at 800,000 frames per second. Individual animals flowing through the light-sheet at 1 meter per second were imaged in 1 millisecond, and populations of hundreds were imaged in seconds. The cytometer has an average resolution of ~3.5 μm in each dimension, with a signal-to-noise ratio over 20 when imaging C. elegans. The system’s potential for high-throughput drug screening was demonstrated by imaging thousands of polyglutamine-mediated aggregation model C. elegans. Statistically-significant phenotypic differences between healthy and unhealthy animals after drug treatment were identified in under a second. Future LEAD cytometers can reach higher resolutions and field-of-views without sacrificing frame rate. LEAD microscopy was then developed for two-photon imaging of brain activity. A galvanometric mirror scans a ~1×22 μm² line across the field-of-view at 1,300 Hz, and the photomultiplier tube array images the line as it scans to reach 2,600 frames per second. Fluorescent beads are imaged with a lateral resolution of 1 – 2 μm. The incorporation of temporal focusing enables an axial resolution of ~5 μm, and reduces out-of-focus fluorescence. A higher-speed two-photon LEAD microscope with acousto-optic scanning at up to 200,000 frames per second and 400 volumes per second is designed for high-speed imaging of action potentials

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