Serial block−face scanning electron microscopy to reconstruct three−dimensional tissue nanostructure
Winfried Denk,Heinz Horstmann +1 more
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It is demonstrated that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope, opening the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.Abstract:
Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H2O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.read more
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Structural and molecular interrogation of intact biological systems
Kwanghun Chung,Jenelle Wallace,Sung-Yon Kim,Sandhiya Kalyanasundaram,Aaron S. Andalman,Thomas J. Davidson,Julie J. Mirzabekov,Kelly A. Zalocusky,Joanna Mattis,Aleksandra K. Denisin,Sally Pak,Hannah L. Bernstein,Charu Ramakrishnan,Logan Grosenick,Viviana Gradinaru,Karl Deisseroth +15 more
TL;DR: It is shown that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.
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Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system
Jean Livet,Tamily A. Weissman,Hyuno Kang,Ju Lu,Robyn A Bennis,Joshua R. Sanes,Jeff W. Lichtman +6 more
TL;DR: Strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours are presented and may facilitate the analysis of neuronal circuitry on a large scale.
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Highly nonrandom features of synaptic connectivity in local cortical circuits.
Sen Song,Per Jesper Sjöström,Per Jesper Sjöström,Markus Reigl,Sacha B. Nelson,Dmitri B. Chklovskii +5 more
TL;DR: The local cortical network structure can be viewed as a skeleton of stronger connections in a sea of weaker ones, likely to play an important role in network dynamics and should be investigated further.
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Reconstruction and Simulation of Neocortical Microcircuitry
Henry Markram,Henry Markram,Eilif Muller,Srikanth Ramaswamy,Michael W. Reimann,Marwan Abdellah,Carlos Aguado Sanchez,Anastasia Ailamaki,Lidia Alonso-Nanclares,Lidia Alonso-Nanclares,Nicolas Antille,Selim Arsever,Guy Antoine Atenekeng Kahou,Thomas K. Berger,Ahmet Bilgili,Nenad Buncic,Athanassia Chalimourda,Giuseppe Chindemi,Jean Denis Courcol,Fabien Delalondre,Vincent Delattre,Shaul Druckmann,Shaul Druckmann,Raphael Dumusc,James Dynes,Stefan Eilemann,Eyal Gal,Michael Gevaert,Jean Pierre Ghobril,Albert Gidon,Joe W. Graham,Anirudh Gupta,Valentin Haenel,Etay Hay,Thomas Heinis,Thomas Heinis,Juan Hernando,Michael L. Hines,Lida Kanari,Daniel Keller,John Kenyon,Georges Khazen,Yihwa Kim,James G. King,Zoltán F. Kisvárday,Pramod Kumbhar,Sebastien Lasserre,Jean Vincent Le Bé,Bruno R. C. Magalhães,Angel Merchán-Pérez,Angel Merchán-Pérez,Julie Meystre,Benjamin Roy Morrice,Jeffrey Muller,Alberto Muñoz-Céspedes,Alberto Muñoz-Céspedes,Shruti Muralidhar,Keerthan Muthurasa,Daniel Nachbaur,Taylor Howard Newton,Max Nolte,Aleksandr Ovcharenko,Juan Palacios,Luis Pastor,Rodrigo Perin,Rajnish Ranjan,Rajnish Ranjan,Imad Riachi,José-Rodrigo Rodríguez,José-Rodrigo Rodríguez,Juan Luis Riquelme,Christian Rössert,Konstantinos Sfyrakis,Ying Shi,Ying Shi,Julian C. Shillcock,Gilad Silberberg,Ricardo Silva,Farhan Tauheed,Martin Telefont,Maria Toledo-Rodriguez,Thomas Tränkler,Werner Van Geit,Jafet Villafranca Diaz,Richard Walker,Yun Wang,Yun Wang,Stefano M. Zaninetta,Javier DeFelipe,Javier DeFelipe,Sean Hill,Idan Segev,Felix Schürmann +92 more
TL;DR: A first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat is presented, finding a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms.
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Imaging Large-Scale Neural Activity with Cellular Resolution in Awake, Mobile Mice
TL;DR: A technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice with minimal motion artifact is reported, demonstrating that running-associated brain motion is limited to approximately 2-5 microm.
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