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Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)(7, 5, 9), de...

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Autores principales: Choe, Yoonsuck, Mayerich, David, Kwon, Jaerock, Miller, Daniel E., Sung, Chul, Chung, Ji Ryang, Huffman, Todd, Keyser, John, Abbott, Louise C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369654/
https://www.ncbi.nlm.nih.gov/pubmed/22215068
http://dx.doi.org/10.3791/3248
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author Choe, Yoonsuck
Mayerich, David
Kwon, Jaerock
Miller, Daniel E.
Sung, Chul
Chung, Ji Ryang
Huffman, Todd
Keyser, John
Abbott, Louise C.
author_facet Choe, Yoonsuck
Mayerich, David
Kwon, Jaerock
Miller, Daniel E.
Sung, Chul
Chung, Ji Ryang
Huffman, Todd
Keyser, John
Abbott, Louise C.
author_sort Choe, Yoonsuck
collection PubMed
description Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)(7, 5, 9), developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)(1, 4, 8), vascular networks (India ink)(1, 4), and cell body distribution (Nissl)(3). The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain(6), mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research(10); to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.
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spelling pubmed-33696542012-06-08 Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy Choe, Yoonsuck Mayerich, David Kwon, Jaerock Miller, Daniel E. Sung, Chul Chung, Ji Ryang Huffman, Todd Keyser, John Abbott, Louise C. J Vis Exp Bioengineering Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)(7, 5, 9), developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)(1, 4, 8), vascular networks (India ink)(1, 4), and cell body distribution (Nissl)(3). The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain(6), mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research(10); to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization. MyJove Corporation 2011-12-09 /pmc/articles/PMC3369654/ /pubmed/22215068 http://dx.doi.org/10.3791/3248 Text en Copyright © 2011, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc/2.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits non-commercial use, distribution, and reproduction, provided the original work is properly cited.
spellingShingle Bioengineering
Choe, Yoonsuck
Mayerich, David
Kwon, Jaerock
Miller, Daniel E.
Sung, Chul
Chung, Ji Ryang
Huffman, Todd
Keyser, John
Abbott, Louise C.
Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title_full Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title_fullStr Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title_full_unstemmed Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title_short Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
title_sort specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369654/
https://www.ncbi.nlm.nih.gov/pubmed/22215068
http://dx.doi.org/10.3791/3248
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