7 research outputs found
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Development of swept, confocally-aligned planar excitation (SCAPE) microscopy for high-speed, volumetric imaging of biological tissue
With the wide-spread adoption of exogenous fluorescent indicators – and more recently genetically encoded fluorescent proteins – over the past two decades, there exists a diverse chemical toolkit with which to probe biological systems. Individual cell types and sub-cellular compartments can be targeted in an increasingly wide range of model organisms. However, imaging these samples is often an exercise in balancing the needs of any given experiment against the constraints of the chosen imaging technology. For example, a volume of brain tissue is host to neurons, glia, vascular compartments and red blood cells that all occupy discrete locations in 3D space, but must work together to support healthy organ function. Single-cell activity on the order of milliseconds can trigger downstream processes that unfold over the course of multiple seconds or even minutes. The development of a technique capable of providing depth-resolved, volumetric imaging with scalable spatiotemporal resolution is crucial to developing a proper understanding of such biological systems.
Bottlenecks in the throughput of existing technologies stem from a combination of inefficient illumination and volume acquisition strategies, and insufficient sensor read-out speeds. Light sheet microscopy is a promising solution, but individual designs tend to be highly specialized to specific types of samples and do not easily adapt to a wide range of experimental settings. In this thesis, I detail my work in developing swept, confocally-aligned planar excitation (SCAPE) microscopy from a first-generation prototype into a versatile, easy-to-reproduce, easy-to-use system for high-speed, 3D imaging.
The first chapter introduces the challenges of designing optical systems capable of high-speed, volumetric imaging. An introduction to design choices faced in the construction of fluorescence microscopes, and current approaches to 3D imaging are discussed. The second chapter describes the progression from the 1st to 2nd generation SCAPE system. Improvements made through ray-tracing models and an enhanced optomechanical design are described, and results from this system in a number of model organisms are presented. The third chapter presents results from a range of biological applications to which SCAPE microscopy has been applied. Work in imaging the zebrafish heart to demonstrate the system’s improved imaging speed, the C. elegans to show the system’s resolution, and finally a number of examples of large field-of-view and high-resolution structural imaging are all described. Finally, the fourth chapter concludes with an overview of the work that lies ahead to both further develop of SCAPE microscopy, as well as to bring the existing system’s strengths to bear in a wider range of environments
Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization
Across the nervous system, neurons with similar attributes are topographically organized. This topography reflects developmental pressures. Oddly, vestibular (balance) nuclei are thought to be disorganized. By measuring activity in birthdated neurons, we revealed a functional map within the central vestibular projection nucleus that stabilizes gaze in the larval zebrafish. We first discovered that both somatic position and stimulus selectivity follow projection neuron birthdate. Next, with electron microscopy and loss-of-function assays, we found that patterns of peripheral innervation to projection neurons were similarly organized by birthdate. Finally, birthdate revealed spatial patterns of axonal arborization and synapse formation to projection neuron outputs. Collectively, we find that development reveals previously hidden organization to the input, processing, and output layers of a highly conserved vertebrate sensorimotor circuit. The spatial and temporal attributes we uncover constrain the developmental mechanisms that may specify the fate, function, and organization of vestibulo-ocular reflex neurons. More broadly, our data suggest that, like invertebrates, temporal mechanisms may assemble vertebrate sensorimotor architecture
Audiovisualization of real-time neuroimaging data.
Advancements in brain imaging techniques have significantly expanded the size and complexity of real-time neuroimaging and behavioral data. However, identifying patterns, trends and synchronies within these datasets presents a significant computational challenge. Here, we demonstrate an approach that can translate time-varying neuroimaging data into unique audiovisualizations consisting of audible representations of dynamic data merged with simplified, color-coded movies of spatial components and behavioral recordings. Multiple variables can be encoded as different musical instruments, letting the observer differentiate and track multiple dynamic parameters in parallel. This representation enables intuitive assimilation of these datasets for behavioral correlates and spatiotemporal features such as patterns, rhythms and motifs that could be difficult to detect through conventional data interrogation methods. These audiovisual representations provide a novel perception of the organization and patterns of real-time activity in the brain, and offer an intuitive and compelling method for complex data visualization for a wider range of applications
3DeeCellTracker, a deep learning-based pipeline for segmenting and tracking cells in 3D time lapse images
Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and ‘straightened’ freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90–100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze
The spatial and temporal structure of neural activity across the fly brain
Abstract What are the spatial and temporal scales of brainwide neuronal activity? We used swept, confocally-aligned planar excitation (SCAPE) microscopy to image all cells in a large volume of the brain of adult Drosophila with high spatiotemporal resolution while flies engaged in a variety of spontaneous behaviors. This revealed neural representations of behavior on multiple spatial and temporal scales. The activity of most neurons correlated (or anticorrelated) with running and flailing over timescales that ranged from seconds to a minute. Grooming elicited a weaker global response. Significant residual activity not directly correlated with behavior was high dimensional and reflected the activity of small clusters of spatially organized neurons that may correspond to genetically defined cell types. These clusters participate in the global dynamics, indicating that neural activity reflects a combination of local and broadly distributed components. This suggests that microcircuits with highly specified functions are provided with knowledge of the larger context in which they operate