Intelligent image-based in situ single-cell isolation

Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.Peer reviewe

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties

Postsynaptic protein organization revealed by electron microscopy.

Neuronal synapses are key devices for transmitting and processing information in the nervous system. Synaptic plasticity, generally regarded as the cellular basis of learning and memory, involves changes of subcellular structures that take place at the nanoscale. High-resolution imaging methods, especially electron microscopy (EM), have allowed for quantitative analysis of such nanoscale structures in different types of synapses. In particular, the semi-ordered organization of neurotransmitter receptors and their interacting scaffolds in the postsynaptic density have been characterized for both excitatory and inhibitory synapses by studies using various EM techniques such as immuno-EM, electron tomography of high-pressure freezing and freeze-substituted samples, and cryo electron tomography. These techniques, in combination with new correlative approaches, will further facilitate our understanding of the molecular organization underlying diverse functions of neuronal synapses

Dense 4D nanoscale reconstruction of living brain tissue

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue

Cross‑Modal Reaction of Auditory and Visual Cortices After Long‑Term Bilateral Hearing Deprivation in the Rat

[Abstract] Visual cortex (VC) over-activation analysed by evoked responses has been demonstrated in congenital deafness and after longterm acquired hearing loss in humans. However, permanent hearing deprivation has not yet been explored in animal models. Thus, the present study aimed to examine functional and molecular changes underlying the visual and auditory cross-modal reaction. For such purpose, we analysed cortical visual evoked potentials (VEPs) and the gene expression (RT-qPCR) of a set of markers for neuronal activation (c-Fos) and activity-dependent homeostatic compensation (Arc/Arg3.1). To determine the state of excitation and inhibition, we performed RT-qPCR and quantitative immunocytochemistry for excitatory (receptor subunits GluA2/3) and inhibitory (GABAA-α1, GABAB-R2, GAD65/67 and parvalbumin-PV) markers. VC over-activation was demonstrated by a signifcant increase in VEPs wave N1 and by up-regulation of the activity-dependent early genes c-Fos and Arc/Arg3.1 (thus confrming, by RT-qPCR, our previously published immunocytochemical results). GluA2 gene and protein expression were signifcantly increased in the auditory cortex (AC), particularly in layers 2/3 pyramidal neurons, but inhibitory markers (GAD65/67 and PV-GABA interneurons) were also signifcantly upregulated in the AC, indicating a concurrent increase in inhibition. Therefore, after permanent hearing loss in the rat, the VC is not only over-activated but also potentially balanced by homeostatic regulation, while excitatory and inhibitory markers remain imbalanced in the AC, most likely resulting from changes in horizontal intermodal regulationMinisterio de Economía y Competitividad; SAF2016–78898-C2-2-RMinisterio de Economía y Competitividad; BFU2017-82375-RJunta de Castilla y León; SA070P1

Osteotomies in Orthognathic Surgery

Orthognathic surgery is mostly performed to correct developmental or acquired oral and maxillofacial skeletal deformities (OMSDs). During the past three decades, significant advances in surgical osteotomy techniques and instrumentation have been developed and carried out in orthognathic surgery. However, the basic surgical principles have more or less remained unchanged. At the same time, numerous surgical techniques have been developed and refined and used by surgeons in the field of oral and maxillofacial surgery. These techniques have treatment of the most complex dentofacial deformities with confidence. Additionally, it has been possible to predict the results of the treatment. Although the initial surgical techniques for correction of anterior mandibular open bite were reported as early as the late 1800s, widespread use of currently acceptable techniques began in the middle of the last century. Detailed surgical planning is essential for a successful outcome. The treatment involves an accurate treatment plan, correct type of instruments for a specific procedure, a thorough surgical routine, and adherence to the guidelines for each routine. Although similar orthognathic surgical techniques are used, there are multiple important differences related to each osteotomy. It is essential for the surgeon to understand these differences in order to provide an effective and safe surgical care for the patient with facial anomalies. Choosing an optimal method of osteotomy depends on many factors, including the indication for treatment, the goal of therapy, patient profile, underlying medical conditions, and the magnitude of surgical movement. The major objective of this chapter is to provide practical guidelines and principles of osteotomies and commonly used techniques. These guidelines are based on a review of the current literature and the author\u27s personal experience. The chapter focuses on the history of orthognathic surgery, anatomical considerations, indications for different osteotomies, and the surgical technique for each osteotomy. Techniques such as the Le Fort I, II, III osteotomies, segmental osteotomies of the maxilla, bilateral sagittal split osteotomy (BSSO), bilateral vertical osteotomy (BVO) genioplasty, segmental osteotomy of the mandible, and the chin wing osteotomy are described

Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Li, S., Raychaudhuri, S., Lee, S. A., Brockmann, M. M., Wang, J., Kusick, G., Prater, C., Syed, S., Falahati, H., Ramos, R., Bartol, T. M., Hosy, E., & Watanabe, S. Asynchronous release sites align with NMDA receptors in mouse hippocampal synapses. Nature Communications, 12(1), (2021): 677, https://doi.org/10.1038/s41467-021-21004-x.Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently.e also thank the Marine Biological Laboratory and their Neurobiology course for supporting the initial set of experiments (course supported by National Institutes of Health grant R25NS063307). S.W. and this work were supported by start-up funds from the Johns Hopkins University School of Medicine, Johns Hopkins Discovery funds, and the National Science Foundation (1727260), the National Institutes of Health (1DP2 NS111133-01 and 1R01 NS105810-01A1) awarded to S.W. S.W. is an Alfred P. Sloan fellow, McKnight Foundation Scholar, and Klingenstein and Simons Foundation scholar. G.K. was supported by a grant from the National Institutes of Health to the Biochemistry, Cellular and Molecular Biology Program of the Johns Hopkins University School of Medicine (T32 GM007445) and is a National Science Foundation Graduate Research Fellow (2016217537). E.H. and T.M.B. are supported by CRCNS-NIH-ANR grant AMPAR-T. The EM ICE high-pressure freezer was purchased partly with funds from an equipment grant from the National Institutes of Health (S10RR026445) awarded to Scot C Kuo

Use of Coherent Point Drift in computer vision applications

This thesis presents the novel use of Coherent Point Drift in improving the robustness of a number of computer vision applications. CPD approach includes two methods for registering two images - rigid and non-rigid point set approaches which are based on the transformation model used. The key characteristic of a rigid transformation is that the distance between points is preserved, which means it can be used in the presence of translation, rotation, and scaling. Non-rigid transformations - or affine transforms - provide the opportunity of registering under non-uniform scaling and skew. The idea is to move one point set coherently to align with the second point set. The CPD method finds both the non-rigid transformation and the correspondence distance between two point sets at the same time without having to use a-priori declaration of the transformation model used. The first part of this thesis is focused on speaker identification in video conferencing. A real-time, audio-coupled video based approach is presented, which focuses more on the video analysis side, rather than the audio analysis that is known to be prone to errors. CPD is effectively utilised for lip movement detection and a temporal face detection approach is used to minimise false positives if face detection algorithm fails to perform. The second part of the thesis is focused on multi-exposure and multi-focus image fusion with compensation for camera shake. Scale Invariant Feature Transforms (SIFT) are first used to detect keypoints in images being fused. Subsequently this point set is reduced to remove outliers, using RANSAC (RANdom Sample Consensus) and finally the point sets are registered using CPD with non-rigid transformations. The registered images are then fused with a Contourlet based image fusion algorithm that makes use of a novel alpha blending and filtering technique to minimise artefacts. The thesis evaluates the performance of the algorithm in comparison to a number of state-of-the-art approaches, including the key commercial products available in the market at present, showing significantly improved subjective quality in the fused images. The final part of the thesis presents a novel approach to Vehicle Make & Model Recognition in CCTV video footage. CPD is used to effectively remove skew of vehicles detected as CCTV cameras are not specifically configured for the VMMR task and may capture vehicles at different approaching angles. A LESH (Local Energy Shape Histogram) feature based approach is used for vehicle make and model recognition with the novelty that temporal processing is used to improve reliability. A number of further algorithms are used to maximise the reliability of the final outcome. Experimental results are provided to prove that the proposed system demonstrates an accuracy in excess of 95% when tested on real CCTV footage with no prior camera calibration