この作品はグラント NFSC 81170806 によって部分的に支えられました。著者は、スキャンやラットの大腿骨を分析支援のための武漢大学口腔病学のマイクロ CT コア施設に感謝したいと思います。

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	<li>Ruegsegger, P., Koller, B., Muller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtomographic+system+for+the+nondestructive+evaluation+of+bone+architecture.">A microtomographic system for the nondestructive evaluation of bone architecture.</a> Calcif Tissue Int. 58 (1), 24-29 (1996).</li><li>Muller, R., Ruegsegger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-tomographic+imaging+for+the+nondestructive+evaluation+of+trabecular+bone+architecture.">Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture.</a> Stud Health Technol Inform. 40, 61-79 (1997).</li><li>Clark, D. P., Badea, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+of+rodents:+state-of-the-art+and+future+perspectives.">Micro-CT of rodents: state-of-the-art and future perspectives.</a> Phys Med. 30 (6), 619-634 (2014).</li><li>Bart, Z., Wallace, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcomputed+Tomography+Applications+in+Bone+and+Mineral+Research.">Microcomputed Tomography Applications in Bone and Mineral Research.</a> Advances in Computed Tomography. 2, 121-127 (2013).</li><li>Ji, Y., Ke, Y., Gao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+activation+of+notch+signaling+promotes+bone+formation.">Intermittent activation of notch signaling promotes bone formation.</a> Am J Transl Res. 9 (6), 2933-2944 (2017).</li><li>Jiang, Y., Zhao, J., White, D. L., Genant, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro+CT+and+Micro+MR+imaging+of+3D+architecture+of+animal+skeleton.">Micro CT and Micro MR imaging of 3D architecture of animal skeleton.</a> J Musculoskelet Neuronal Interact. 1 (1), 45-51 (2000).</li><li>Laib, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+micro-computed+tomography+of+trabecular+and+cortical+bone+architecture+with+application+to+a+rat+model+of+immobilisation+osteoporosis.">3D micro-computed tomography of trabecular and cortical bone architecture with application to a rat model of immobilisation osteoporosis.</a> Med Biol Eng Comput. 38 (3), 326-332 (2000).</li><li>Cole, H. A., Ichikawa, J., Colvin, D. C., O'Rear, L., Schoenecker, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+intra-osseous+growth+of+osteosarcoma+in+a+murine+model+with+radiographic+analysis.">Quantifying intra-osseous growth of osteosarcoma in a murine model with radiographic analysis.</a> J Orthop Res. 29 (12), 1957-1962 (2011).</li><li>Jensen, M. M., Jorgensen, J. T., Binderup, T., Kjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+subcutaneous+mouse+xenografts+measured+by+microCT+is+more+accurate+and+reproducible+than+determined+by+18F-FDG-microPET+or+external+caliper.">Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper.</a> BMC Med Imaging. 8, 16 (2008).</li><li>Soviero, V. M., Leal, S. C., Silva, R. C., Azevedo, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+MicroCT+for+in+vitro+detection+of+proximal+carious+lesions+in+primary+molars.">Validity of MicroCT for in vitro detection of proximal carious lesions in primary molars.</a> J Dent. 40 (1), 35-40 (2012).</li><li>Kohler, T., Stauber, M., Donahue, L. R., Muller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+compartmental+analysis+for+high-throughput+skeletal+phenotyping+in+femora+of+genetic+mouse+models.">Automated compartmental analysis for high-throughput skeletal phenotyping in femora of genetic mouse models.</a> Bone. 41 (4), 659-667 (2007).</li><li>Buie, H. R., Campbell, G. M., Klinck, R. J., MacNeil, J. A., Boyd, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+segmentation+of+cortical+and+trabecular+compartments+based+on+a+dual+threshold+technique+for+in+vivo+micro-CT+bone+analysis.">Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis.</a> Bone. 41 (4), 505-515 (2007).</li><li>Dougherty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+CT+in+the+measurement+of+bone+quantity+and+bone+quality+for+assessing+osteoporosis.">Quantitative CT in the measurement of bone quantity and bone quality for assessing osteoporosis.</a> Med Eng Phys. 18 (7), 557-568 (1996).</li><li>Doube, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BoneJ:+Free+and+extensible+bone+image+analysis+in+ImageJ.">BoneJ: Free and extensible bone image analysis in ImageJ.</a> Bone. 47 (6), 1076-1079 (2010).</li><li>Bouxsein, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Miner Res. 25 (7), 1468-1486 (2010).</li></ol>

オブジェクトの厚さの定量化の基になるアルゴリズムの特許が提出されています。著者と共同で他の人は bomomics.com ウェブサイトのコンサルティングと定量化画像解析サービスをリクエストに応じて提供する、無料版プラグインをホスティングを登録します。

本研究では、海綿骨部、自動、効率的であるとユーザー ・ フレンドリーを分析するための ImageJ プラグインについて説明します。任意の 2D または 3D オブジェクト領域、ボリューム、および厚さの層によって対策を定量化するプラグインが使えます。現在、骨梁パラメーターごとに単一の測定値のみは、各サンプルの選択した分析領域で測定されたエンティティの特性を表すことはできませ?...

海綿骨のマイクロ CT 解析は、異なる骨損失条件1,2,3、いくつかの変数が関連の下で小動物の骨の形態学的変化を追跡するための標準的なアプローチ、骨の構造は、報告された4です。しかし、このようなパラメーターが5長骨の骨幹端に均等に配布されていない、現在商用マイクロ CT 機6,7 から各サンプルの各構造変数の要約または平均値のみが報告されます。、単一の値は完全に分析領域の測定パラメーターの特性を表すことはできません。海綿骨の層によって定量化だけでなく各変数の詳細を保持、また構造の後の統計的分析を促進する分析地域のような変数の分布のプロファイリングを有効に5さまざまな条件下での変更。したがって、このメソッドの目的は、現在、市販のマイクロ CT 解析パッケージで利用されていません、各スライス レベルでマイクロ CT スキャンの海綿骨を定量化は。
効率的にセグメント海綿骨--スライスごとに自動分割方法が望ましいです。ただし、マイクロ CT 解析のための現在の標準的な技術、労働集約的でエラーを起こしやすい、骨皮質のコンパートメントから分離する半自動補間続いて手動対話型輪郭に基づいて、実質的な演算子バイアス8,9,10に関連付けられています。自動セグメンテーション方法11,12は報告されたが、海綿骨と皮質骨の間良好な分離がある地域で地域の明確な分離なしではなく、このようなメソッドは最適です。さらに、異なるセグメンテーション パラメーターは異なるサンプル12、必須、様々 なパラメーターの組み合わせ12を試みることによって骨のサンプルのグループに適用される満足な分割パラメーターを手動で選択するは面倒です。にもかかわらず、すべての関連するパラメーターが設定されている場合、分割プロセスは自動です。骨の外側の境界がスキャンの背景を持つ最大のコントラストと長骨の骨幹端部皮質シェルは、選択した領域を分析ことができます長い骨の外側輪郭に応じてセグメンテーション方法いくつかの変更を表示確実に、正確には、皮質の殻から海綿骨を区切ります。このような分割方法の利点は、セグメンテーションは海綿骨と皮質骨6,12の違いではなく背景と骨の外側の境界の違いに基づいています。 13, したがって、それは一般的に異なるグループの間の骨梁変化のより信頼性の高い解析を促進する骨のサンプルのグループの満足できる分割パラメーターの組み合わせを見つけるは簡単。
各スライスにはボリューム、サーフェス、および 3 次元 (3 D) の厚さは 3 D 数量で報告される間、レベル、エリア、境界、および二次元 (2 D) 厚さが 2D 解析の報告されます。そのような情報は、一般的に現在画像解析ツールは、そのような情報が必要な一般のイメージに報告された手順を適用できることを示すから報告されません。

<table><tbody><tr><td>ImageJ</td><td>NIH</td><td>imagej</td><td>Any version with a java 1.8 run time</td></tr><tr><td>trabecular analysis plugin</td><td>Bomomics</td><td>bomomics</td><td>free or commercial version</td></tr><tr><td>Micro CT scanner</td><td>Scanco</td><td>&amp;mu;-50</td><td>micro CT from any vendor</td></tr><tr><td>Computer System</td><td>Lenovo</td><td></td><td>any brand</td></tr><tr><td>Windows Operating System</td><td>Microsoft</td><td>Windows 7 x64</td><td>any 64-bit Windows operating system&amp;nbsp;</td></tr><tr><td>Office Software</td><td>Microsoft</td><td>Office 2010</td><td>any speadsheet software that has xy chart function</td></tr></tbody></table>

outer-boundary assisted segmentation and quantification of trabecular bones by an imagej plugin

マイクロ コンピューター断層撮影 (マイクロ CT) は、定期的に骨量と異なる骨損失条件下で小動物の骨梁の微細構造プロパティを評価するために使用されます。マイクロ CT 画像の骨の分析のための標準的なアプローチは、スライスによって半自動手-輪郭、労働集約的、間違いやすい作業であります。骨の外側の境界、海綿骨を識別して少ないオペレーター バイアスとき適切な精度で自動的に分割によると海綿骨の自動セグメンテーションのための効率的な方法は、ここで説明しました。分割パラメーターが設定されます。分割パラメーターのすべての組み合わせが変更された 1 つずつ順番に、関連するパラメーターと領域分割結果が満足のいくセグメンテーション パラメーターをプロファイルに領域分割結果の画像のスタックが表示されます。簡単に視覚的に確認します。プラグインの品質管理機能として理論値と測定量を比較することができます, シミュレートされた標準的なオブジェクトの量を示します。海綿の性質、骨梁の厚さの層によって定量化がこのようなプラグインによって報告され、選択した領域内のような特性の分布を簡単にプロファイリングできます。措置がここで一つだけ現在の商用ソフトウェアの出力から利用できない層によって定量化海綿骨に関するより多くの情報を保持し、構造変化の統計的分析が容易にさらに、各パラメーターの定量化された値は、サンプルごとに報告されます。したがって、記載されているワークフローは、精度と効率による海綿骨の分析により良いアプローチです。

骨梁の解析プラグインは自動的にセグメント化し、精度と海綿骨を定量化する設計されています。当初、骨の外側の境界が検出され、線引き後に穴充填操作骨外側皮質シェル内の任意の穴が満ちています。その後、外側の皮質骨を除外し、セグメント化された海綿骨を得る侵食操作を実行します。最後に、海綿骨部のセグメント化された地域の対策は明らかにした.</p...

Watch this Scientific Journal Video about Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin at JoVE.com

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

セグメント化および定量化 ImageJ プラグインを使用して骨の外側の境界に基づく 2 D と 3 D 画像の海綿骨のワークフローを提案します。このアプローチはより効率的かつ現在の手動手輪郭アプローチよりも正確な現在の商用ソフトウェアでは利用できないする層によって数量を提供しています。

外側境界セグメンテーションと Imagej のプラグインで海綿骨の定量化を支援

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

outer-boundary-assisted-segmentation-quantification-trabecular-bones

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.2K ビュー.  Wuhan University. セグメント化および定量化 ImageJ プラグインを使用して骨の外側の境界に基づく 2 D と 3 D 画像の海綿骨のワークフローを提案します。このアプローチはより効率的かつ現在の手動手輪郭アプローチよりも正確な現在の商用ソフトウェアでは利用できないする層によって数量を提供しています。

ビデオ: Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

発生生物学

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

心房と心室から心筋細胞における管状膜ネットワークの解析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

生物工学

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

がん研究

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

半自動縦マイクロ トモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

心臓トラベキュラEx Vivoの同時ブライトフィールド、蛍光、光コヘレンス断層イメージング

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reliable results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to a lack of programming knowledge and paywalls for software. In addition, cells of interest display movements in all directions during calcium imaging. Many approaches have been developed to correct the motion in the lateral (x/y) direction.
This paper describes a workflow using a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI), to examine motion on the z-axis in 3D calcium imaging. This software identifies the maximum fluorescence value from all the z-positions a neuron appears in and uses it to represent the neuron&#39;s intensity at the corresponding t-position. Therefore, this tool can separate neurons overlapping in the lateral (x/y) direction but appearing&#160;on distinct z-planes. As an ImageJ plugin, TACI is a user-friendly, open-source computational tool for 3D calcium imaging analysis. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.

TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes.

TACI: 3D カルシウムイメージング解析のための ImageJ プラグイン

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging ...

神経科学

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

骨関節炎性軟骨下骨厚さのソフトウェア支援定量測定

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

医学

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

生物学

マウス海綿骨の微細構造を評価するためのHE染色とマイクロCT

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

著者スポットライト:マウス骨粗鬆症モデルにおける骨マイクロアーキテクチャの定量的評価のための経済的かつ効率的な方法 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

骨粗鬆症マウスモデルにおける海綿骨マイクロアーキテクチャ評価

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

外側境界セグメンテーションと Imagej のプラグインで海綿骨の定量化を支援

要約

さらに動画を探す

この動画の章

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

心房と心室から心筋細胞における管状膜ネットワークの解析

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

半自動縦マイクロトモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

心臓トラベキュラEx Vivoの同時ブライトフィールド、蛍光、光コヘレンス断層イメージング

TACI: 3D カルシウムイメージング解析のための ImageJ プラグイン

骨関節炎性軟骨下骨厚さのソフトウェア支援定量測定

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

骨粗鬆症マウスモデルにおける海綿骨マイクロアーキテクチャ評価

外側境界セグメンテーションと Imagej のプラグインで海綿骨の定量化を支援

要約

さらに動画を探す

この動画の章

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

心房と心室から心筋細胞における管状膜ネットワークの解析

乳腺腫瘍微小環境の生体内イメージングのためのラベルフリーセグメンテーションアプローチ

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

半自動縦マイクロ トモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

心臓トラベキュラEx Vivoの同時ブライトフィールド、蛍光、光コヘレンス断層イメージング

TACI: 3D カルシウムイメージング解析のための ImageJ プラグイン

骨関節炎性軟骨下骨厚さのソフトウェア支援定量測定

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

骨粗鬆症マウスモデルにおける海綿骨マイクロアーキテクチャ評価

半自動縦マイクロトモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折