Prof. Timothy E. Holy Profile

Prof. Timothy E. Holy

at Washington Univ. School of Medicine in St. Louis

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Publications (3)

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Proceedings Article | 5 March 2021 Presentation + Paper

Fast volumetric imaging of neural activity in deep brain

Che-Pin (Jonathan) Chang, Timothy Holy

Proceedings Volume 11629, 116290L (2021) https://doi.org/10.1117/12.2582619

KEYWORDS: Microscopes, Mirrors, Tissue optics, Objectives, Microscopy, Imaging systems, Calcium, Brain, 3D image processing, Tissues

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Calcium imaging is a widely-used technique for recording neuronal activity. For deep-brain imaging, light scattering degrades image acquisition. To avoid imaging through thick tissue, one common approach is to implant a small lens system (a microendoscope) into the brain. But there is no technique to achieve fast volumetric imaging through such lenses, and this lack forces a choice between abandoning optical sectioning or sampling with risk of confusion from overlaps (when labeling is dense) or being limited to modest neural population size (when labeling is sparse). To address these limitations, we designed a novel imaging technique, RE-imaging Axial Light-sheet Microscopy (REALM), suitable for fast three-dimensional imaging through a microendoscope. REALM images via a tilted light-sheet, illuminating and collecting fluorescence emission with single objective. The first-stage “Maxwell theorem” microscope employs a matching pair of objectives to reimage sample volume onto a sawtooth mirror, which consists of a series of sub-micrometer scale angled surfaces. The sawtooth mirror redirects the light horizontally into the second-stage microscope, forming a crisp image of the illuminated near-axial plane. The whole second microscope system collects over 40% of light reflected by the sawtooth mirror, compared to previous studies 28% of light collection efficiency at numerical apertures that are unavailable for microendoscopy. This microscope will combine the speed and resolution advantages of light-sheet microscopy with the capabilities of microendoscopes for deep-brain imaging, providing the ability to perform fast threedimensional imaging in deep tissue.

Proceedings Article | 5 March 2021 Presentation + Paper

Integrated functional characterization and optical tagging for cell-type identification

Donghoon Lee, Maiko Kume, Timothy Holy

Proceedings Volume 11663, 116630I (2021) https://doi.org/10.1117/12.2583433

KEYWORDS: Integration, Integrated optics, Neurons, Visualization, Visual analytics, Sensors, Profiling, Computer programming, Calcium, Brain mapping

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Imaging has become one of the most important tools for categorizing neurons based on their function. However, for a cell type identified only by its pattern of activity, the process of identifying molecular markers remains laborious. We developed physiological optical tagging sequencing (PhOTseq), a technique for tagging and expression profiling of cells on the basis of their functional properties. We developed a reporter combining a green calcium indicator (GCaMP) with a photoactivatable red reporter (PAmCherry). When visualizing neuronal activity in such animals, real-time analysis allowed digital selection of cells exhibiting specific activity patterns, and photoactivation was directed specifically to those cells to tag them for later harvesting and analysis. We found that PhOTseq was capable of selecting rare cell types and enriching them by nearly 100-fold. We applied PhOTseq to the challenge of mapping receptor-ligand pairings among pheromone-sensing neurons in mice, and densely mapped the cell types responsible for encoding a specific portion of the sensory world.

Proceedings Article | 10 September 2011 Paper

Development of low-coherence light sheet illumination microscope for fluorescence-free bioimaging

Zhiguang Xu, Timothy Holy

Proceedings Volume 8129, 812908 (2011) https://doi.org/10.1117/12.893569

KEYWORDS: Tissues, Microscopes, Coherence (optics), Tissue optics, Microscopy, Glasses, Inspection, Light sources, Collimation, Objectives

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Light Sheet Illumination Microscopy (LSIM) is an imaging modality featuring the novel arrangement with the illumination axis perpendicular to the detection axis. In this technology a well defined light sheet is generated and aligned precisely to the focal plane of the microscope objective and thus only the thin in-focus layer of the sample is illuminated and imaged, thereby avoiding out-of-focus light. Besides the inherent optical sectioning function, other advantages include fast imaging speed, high longitudinal resolution and decreased light-induced damage. Though promising, this microscopy is currently restricted to imaging fluorescently labeled tissue; in inspection of intact tissue using scattered light, the acquired images suffer from intense speckles because of the severe coherence in the illumination. This work aims to build a microscope capable of achieving intrinsic images of the fluorescence-free sample with reduced or eliminated speckles, by developing a low coherence light sheet illumination. To diminish the spatial coherence, the sample is illuminated with tens of independent sub-beams (without inter-coherence) illuminating the FOV (Field Of View) of the microscope with diverse incident angles. The temporal coherence is dramatically reduced by employing a supercontinuum laser with a broad spectrum as the light source. The new microscopy significantly extends the functionality of Light Sheet Illumination Microscopy and will enable many new bioimaging applications.

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