Comprehensive geometric modeling of human cerebral vasculature for quantitative vascular analysis

Thesis: S.M., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.Cataloged from PDF version of thesis.Includes bibliographical references (pages 23-25).In this work we developed a comprehensive and structured geometric model of human cerebral vasculature for quantitative anatomical analysis. We first proposed a general and structured geometric representation of the interconnected vascular network as a framework. We then described an image processing pipeline for the segmentation of vascular anatomy from discrete scalar field images, and applied the pipeline to segment the anatomical structures of cerebral vasculatures from whole brain magnetic resonance angiography (MRA) scans of healthy adult subjects. Next, we employed the proposed geometric representation to generate the comprehensive geometric model of human cerebral vasculature from those segmented anatomies. In the end, we performed quantitative anatomical analysis to the anterior cerebral arteries (ACA) and internal carotid arteries (ICA), and characterized their varying size and tortuosity in the cerebral arterial circulation.by Changyang Linghu.S.M

Spatially organized fluorescent reporters for recording complex biological dynamics in cell population

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, February, 2020Cataloged from PDF of thesis.Includes bibliographical references (pages 161-178).Biological signals, such as the dynamic concentrations of ions, levels of signaling molecules, and activities of protein kinases, interact in complex ways within cells, and can exhibit great cell-to-cell heterogeneity as a function of cell history and state. There is increasing desire to use multiple fluorescent reporters to simultaneously measure multiple biological signals in single cells across cell populations, such as those in the brain. However, due to the diffraction limit of optical imaging, the biological signals recorded from neurons in densely-labeled neural populations in vivo are often mixed with signals from closely passing axons and dendrites from other neurons, resulting in erroneous signaling events and artifactual correlations of measured neural activity. Also, it is not yet possible to simultaneously record any given set of biological signals in single cells, because there are limited sets of corresponding spectrally-orthogonal fluorescent reporters available to date. Even if the fluorescent reporters for all biological signals in all possible colors are developed in the future, the number of biological signals that can be simultaneously recorded are still limited by the number of available optical channels. In this thesis, I address these problems by developing two new technologies, soma-targeted fluorescent calcium indicators and spatially multiplexed imaging.Soma-targeted fluorescent calcium indicators (or 'SomaGCaMPs', the first part of the thesis) are fluorescent reporters of calcium dynamics that are selectively localized at the soma, but not axons and dendrites, of neurons. In vivo optical imaging of SomaGCaMPs in dense neural circuits in mouse and zebrafish brains reported fewer artifactual spikes, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent reporters is a simple and powerful method for high-fidelity population imaging of neural activity in vivo.Spatially multiplexed imaging (the second part of the thesis) enables simultaneous readout of multiple biological signals in single cells from fluorescent reporters regardless of their spectra. This is achieved by clustering reporters into spatially separated 'Signaling Reporter Islands' (or 'SiRIs') via self-assembling protein scaffolds or RNA scaffolds. Using the spatial dimension as an asset, SiRIs may open up the ability to simultaneously image nearly arbitrary numbers of signals within a physiological cascade.by Changyang Linghu.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Scienc

Temporally precise single-cell-resolution optogenetics

© 2017 The Author(s). Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in mouse cortical brain slices with single-cell resolution and <1-ms temporal precision. We used soCoChR to perform connectivity mapping on intact cortical circuits

Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics

© 2020 The Author(s) In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

© 2018 The Author(s). We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator

© 2020 Elsevier Inc. One-photon fluorescent imaging of calcium signals can capture the activity of hundreds of neurons across large fields of view but suffers from crosstalk from neuropil. Shemesh et al. engineer cell-body-targeted variants of fluorescent calcium indicators and show in mice and zebrafish that artifactual spikes and correlations are greatly reduced

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50&nbsp;μm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

© 2018 The Author(s). We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans