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      Real-time volumetric microscopy of in-vivo dynamics and large-scale samples with SCAPE 2.0


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          The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes-per-second and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving C. elegans, and analyze real-time blood flow and calcium dynamics in the beating zebrafish heart. The same system also permits high-throughput structural imaging of mounted, intact, cleared and expanded samples. SCAPE 2.0’s significantly lower photodamage compared to point-scanning techniques is also confirmed. Our results demonstrate that SCAPE 2.0 is a powerful, yet accessible imaging platform for myriad emerging high-speed dynamic and high-throughput volumetric microscopy applications.

          Editor’s summary

          SCAPE 2.0 is a versatile imaging platform that enables real-time 3D microscopy of cellular function and dynamic motion in living organisms at over 100 volumes per second with minimal photodamage, and high-throughput structural imaging in fixed, cleared and expanded samples.

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          Most cited references38

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          Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.

          The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.
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            Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms

            We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.
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              Whole-brain calcium imaging with cellular resolution in freely behavingCaenorhabditis elegans

              The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal's position and orientation. Custom software tracks the 3D position of the animal's head in real time and two feedback loops adjust a motorized stage and objective to keep the animal's head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal's behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.

                Author and article information

                Nat Methods
                Nat. Methods
                Nature methods
                22 October 2019
                27 September 2019
                October 2019
                27 March 2020
                : 16
                : 10
                : 1054-1062
                [1 ]Laboratory for Functional Optical Imaging, Department of Biomedical Engineering, Columbia University, New York, New York, USA
                [2 ]Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, New York, USA
                [3 ]Department of Pediatrics, College of Physicians & Surgeons, Columbia University, New York, New York, USA
                [4 ]Graduate School of Natural Sciences, Nagoya City University, Nagoya, Japan
                [5 ]Center for Advanced Intelligence Project, RIKEN, Tokyo, Japan
                [6 ]Department of Radiology, Columbia University Medical Center and New York-Presbyterian Hospital New York, New York, USA
                Author notes

                Equal contributions

                Author Contributions

                V.V and E.M.C.H conceived of and designed the system. V.V., W.L. 1, K.B.P., C.P.C. and E.M.C.H constructed the system. H.Y. and W.L. 2 assisted and provided information for extended designs. V.V, W.L 1, K.B.P, C.P.C, S.B.S, R.W.Y and E.M.C.H performed the experiments. V.V, K.B.P, S.B.S, R.W.Y, M.J.C and E.M.C.H. analyzed data. C.F and K.T created the zebrafish lines and performed husbandry. C.W and K.K. developed the deep-learning tracking algorithm used for cardiomyocyte nuclear tracking and tracked the cells. V.V. and E.M.C.H. wrote and prepared the manuscript. All authors reviewed, edited and consulted on the manuscript text. 1Wenze Li, 2Wenxuan Liang.

                [* ]Corresponding Author: Hillman, Elizabeth MC ( Elizabeth.hillman@ 123456columbia.edu )
                Author information

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                Life sciences
                Life sciences


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