Name: quantitative approaches for studying cellular structures and organelle morphology in caenorhabditis elegans
Uploaded: 2019-07-05T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans at JoVE.com

我们感谢 Neumann 实验室的成员进行了宝贵的讨论和投入。一些菌株由CGC提供,由NIH研究基础设施项目办公室(P40 OD010440)资助。作者感谢WormBase提供了丰富的关于C.elegans的信息,并感谢莫纳什大学的莫纳什微成像提供了仪器、培训和技术支持。这项工作得到了CMTAA研究资助(2015年和2018年)的支持,NHMRC项目赠款1101974和1099690授予了B.N.

1. C. 埃莱甘菌株的生长与维持
<ol><li>种子线虫生长介质 (NGM, 参见材料表) 琼脂板与300 μL的缓慢生长的大肠杆菌菌株OP50在层流柜。</li>
	<li>将 NGM 琼脂板留在层流柜中晾干。 注:在没有层流柜的情况下,板可以留在长凳上干燥,但更容易受到污染。</li>
	<li>将至少20只动物转移到两个OP50种子NGM琼脂板中,以有工作种群。有两种主要的方式来转移动物。<ol>
<li>采摘:使用热消毒?...

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. , (1974).</li><li>Markaki, M., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+in+Caenorhabditis+elegans.">Modeling human diseases in Caenorhabditis elegans.</a> Biotechnology Journal. 5 (12), 1261-1276 (2010).</li><li>Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+swim+sessions+in+C.+elegans+induce+key+features+of+mammalian+exercise.">Single swim sessions in C. elegans induce key features of mammalian exercise.</a> BMC Biology. , (2017).</li><li>Alexander, A. G., Marfil, V., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+C.+elegans+as+a+model+to+study+Alzheimer's+disease+and+other+neurodegenerative+diseases.">Use of C. elegans as a model to study Alzheimer's disease and other neurodegenerative diseases.</a> Frontiers in Genetics. , (2014).</li><li>Zheng, C., Jin, F. Q., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+Proteins+Act+as+Transcriptional+Guarantors+to+Ensure+Terminal+Differentiation.">Hox Proteins Act as Transcriptional Guarantors to Ensure Terminal Differentiation.</a> Cell Reports. 13 (7), 1343-1352 (2015).</li><li>Koushika, S. P., 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=Mutations+in+Caenorhabditis+elegans+cytoplasmic+dynein+components+reveal+specificity+of+neuronal+retrograde+cargo.">Mutations in Caenorhabditis elegans cytoplasmic dynein components reveal specificity of neuronal retrograde cargo.</a> Journal of Neuroscience. 24 (16), 3907-3916 (2004).</li><li>Byrne, J. J., 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=Disruption+of+mitochondrial+dynamics+affects+behaviour+and+lifespan+in+Caenorhabditis+elegans.">Disruption of mitochondrial dynamics affects behaviour and lifespan in Caenorhabditis elegans.</a> Cellular and Molecular Life Sciences. , (2019).</li><li>Campagnola, P. J., 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=Three-Dimensional+High-Resolution+Second-Harmonic+Generation+Imaging+of+Endogenous+Structural+Proteins+in+Biological+Tissues.">Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues.</a> Biophysical Journal. 82 (1), 493-508 (2002).</li><li>Schindelin, J., 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Sommer, C., Straehle, C., Kothe, U., Hamprecht, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ilastik:+Interactive+learning+and+segmentation+toolkit.">Ilastik: Interactive learning and segmentation toolkit.</a> 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. , (2011).</li><li>Mondal, S., Ahlawat, S., Koushika, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+microfluidic+devices+for+in+vivo+imaging+of+C.+elegans,+Drosophila+and+zebrafish.">Simple microfluidic devices for in vivo imaging of C. elegans, Drosophila and zebrafish.</a> Journal of Visualized Experiments. , (2012).</li><li>Paul, G., Cardinale, J., Sbalzarini, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+image+restoration+and+segmentation:+A+generalized+linear+model/bregman+perspective.">Coupling image restoration and segmentation: A generalized linear model/bregman perspective.</a> International Journal of Computer Vision. , (2013).</li><li>Rizk, 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=Segmentation+and+quantification+of+subcellular+structures+in+fluorescence+microscopy+images+using+Squassh.">Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.</a> Nature Protocols. 9, 586-586 (2014).</li><li>Akella, J. S., 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=MEC-17+is+an+alpha-tubulin+acetyltransferase.">MEC-17 is an alpha-tubulin acetyltransferase.</a> Nature. 467 (7312), 218-222 (2010).</li><li>Neumann, B., Hilliard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+MEC-17+leads+to+microtubule+instability+and+axonal+degeneration.">Loss of MEC-17 leads to microtubule instability and axonal degeneration.</a> Cell Reports. 6 (1), 93-103 (2014).</li><li>Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B., Nachury, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+alpha-tubulin+K40+acetyltransferase+alphaTAT1+promotes+rapid+ciliogenesis+and+efficient+mechanosensation.">The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21517-21522 (2010).</li><li>Teoh, J. -. S., Wang, W., Chandhok, G., Pocock, R., Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+maintenance+of+synaptic+structure+is+mediated+by+the+alpha-tubulin+acetyltransferase+MEC-17/&#945;TAT1.">Development and maintenance of synaptic structure is mediated by the alpha-tubulin acetyltransferase MEC-17/&#945;TAT1.</a> bioRxiv. 84, 522904-522904 (2019).</li><li>Bird, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Charcot-Marie-Tooth Neuropathy Type 2. , (1998).</li><li>Chandhok, G., Lazarou, M., Structure Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+regulation+of+mitofusin-2+in+health+and+disease.">Structure, function, and regulation of mitofusin-2 in health and disease.</a> Biological Reviews. 93 (2), 933-949 (2018).</li><li>Soh, M. S., Cheng, X., Liu, J., Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+genes+associated+with+Charcot-Marie-Tooth+type+2+lead+to+common+behavioural,+cellular+and+molecular+defects+in+Caenorhabditis+elegans.">Disruption of genes associated with Charcot-Marie-Tooth type 2 lead to common behavioural, cellular and molecular defects in Caenorhabditis elegans.</a> bioRxiv. , 605584 (2019).</li><li>Kamerkar, S. C., Kraus, F., Sharpe, A. J., Pucadyil, T. J., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-related+protein+1+has+membrane+constricting+and+severing+abilities+sufficient+for+mitochondrial+and+peroxisomal+fission.">Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.</a> Nature Communications. , (2018).</li><li>Zhu, P. -. P., 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=Intra-+and+Intermolecular+Domain+Interactions+of+the+C-terminal+GTPase+Effector+Domain+of+the+Multimeric+Dynamin-like+GTPase+Drp1.">Intra- and Intermolecular Domain Interactions of the C-terminal GTPase Effector Domain of the Multimeric Dynamin-like GTPase Drp1.</a> Journal of Biological Chemistry. 279 (34), 35967-35974 (2004).</li><li>Osellame, L. D., 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=Cooperative+and+independent+roles+of+the+Drp1+adaptors+Mff,+MiD49+and+MiD51+in+mitochondrial+fission.">Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission.</a> Journal of Cell Science. , (2016).</li><li>Dima, A. 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=Comparison+of+segmentation+algorithms+for+fluorescence+microscopy+images+of+cells.">Comparison of segmentation algorithms for fluorescence microscopy images of cells.</a> Cytometry Part A. 79 (7), 545-559 (2011).</li><li>Trevisan, T., 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=Manipulation+of+Mitochondria+Dynamics+Reveals+Separate+Roles+for+Form+and+Function+in+Mitochondria+Distribution.">Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution.</a> Cell Reports. , (2018).</li></ol>

形态变异经常通过手工计算明显差异或使用任意阈值来确定与野生型表型相比的缺陷来评估。然而,最近,定量方法被用于形态学的比较研究,以无偏见的方式准确测量和描述细胞和亚细胞水平上的变化。识别表型之间微妙但与生物学相关差异的能力是了解控制由环境因素或遗传疾病引起的形态变化的基本分子机制的有力手段。计算能力和显微镜成像分辨率的不断提高,加上C. elegans遗传学的易?...

线虫卡埃内沙布迪炎(C.elegans)越来越多地被用作一个模型系统,以揭示人类疾病所涉及的生物和分子过程。成年线虫的体长略高于1毫米,可以产生多达300个鸡蛋的大卵1。孵化后,C.elegans只需3-4天才能成年,并存活2至3周。由于其易于培养,C. elegans是目前最抢手的体内动物模型之一,用于进行经济高效、快速的药物筛选,以确定人类疾病的治疗药物。此外,其遗传保护,明确界定的行为范式,透明的身体荧光或光显微镜,以及易于基因操作,使研究细胞和分子后果的基因突变很容易实现3. C. elegans基因组与人类基因共享大约 60-80% 的正畸,其中约 40% 与疾病相关。在C.elegans中建模和研究的一些人类疾病包括神经退行性疾病(阿尔茨海默氏病、帕金森病、肌萎缩性侧索硬化症、Charcot-Marie-Tooth 病)、肌肉相关疾病(杜琴肌肉萎缩症),和代谢性疾病(高血糖)2,4。在大多数人类疾病中,疾病引起的细胞和细胞器定位和形态变化发生,这很容易在线虫模型中评估。
荧光标记已广泛用于标记组织和细胞器,用于显微镜下的动态可视化。然而,在C.elegans中,评估由于基因突变引起的形态不规则性的传统方法主要依靠视觉描述。虽然定性评估可以涵盖更广泛的表型描述范围(突触形态学、GFP结块、特定斧头形状、肌肉纤维厚度等),并提供形态变化的鸟瞰图,但它们不太适合比较不同组之间的小差异。此外,定性评估以视觉、主观评估为基础,这可能导致对形态异常的高估或低估。最后,定性观测结果也可能因个人而异,给数据复制造成困难。
近年来,已经开发出一些用户友好、现成的计算算法,可以定量分析图像。然而,这种图像分析软件的利用,在一些形态学研究,特别是有关身体壁肌和线粒体的研究,在C.elegans的研究已经滞后。为了改进C.elegans的基本结构分析,试验了一些现成的开源图像分析软件,以定量比较基因突变对肌肉线粒体、身体壁肌和突触的影响形态。这些实验程序详细概述了这些程序(Fiji、ilastik、CellProfiler、SQUASSH)如何用于评估突触大小和突触蛋白定位、体壁肌肉面积和纤维长度以及线粒体尺寸和线粒体尺寸的变化。由于线虫的基因突变而循环。

<table>
	<tbody>
		<tr>
			<td>Agar-agar</td>
			<td>Merck</td>
			<td>1.01614.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td>Inverted platform</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Carl Zeiss AG</td>
			<td>Zeiss Axio Imager M2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips #1</td>
			<td>Thermo scientifique</td>
			<td>MENCS22221GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips #1.5</td>
			<td>Zeiss</td>
			<td>474030-9000-000</td>
			<td>Made by SCHOTT</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermo scientifique</td>
			<td>MENS41104A/40</td>
			<td></td>
		</tr>
		<tr>
			<td>Light LED</td>
			<td>Schott</td>
			<td>KL 300 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone (Peptone from casein)</td>
			<td>Merck</td>
			<td>107213</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck</td>
			<td>103753</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for Lysogeny Broth (LB) medium</td>
		</tr>
		<tr>
			<td>Peptone (Peptone from meat)</td>
			<td>Merck</td>
			<td>107214</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A1296</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>102382</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>105886</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Dipotassium phosphate</td>
			<td>Merck</td>
			<td>105101</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>104873</td>
			<td>Ingredients for Nematode Growth Media (NGM) agar</td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>Merck</td>
			<td>106586</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>104873</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>105886</td>
			<td>Ingredients for M9 buffer</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Corning</td>
			<td>CLS7095D5X-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Corning</td>
			<td>CLS430589-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>Sigma</td>
			<td>267201-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Met-app</td>
			<td>2616</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride</td>
			<td>Sigma</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative approaches for studying cellular structures and organelle morphology in caenorhabditis elegans

定义疾病背后的细胞机制对于开发新的治疗方法至关重要。经常用来解开这些机制的策略是引入候选基因的突变,并定性地描述组织和细胞细胞器的形态变化。然而,定性描述可能无法捕捉细微的型值差异,可能歪曲人群中个体的型型变化,并且经常被主观地评估。本文介绍了定量方法,利用激光扫描共聚焦显微镜与市售的生物图像处理软件相结合,研究线虫卡埃纳哈布迪炎的组织和细胞器的形态。对影响突触完整性(大小和综合荧光水平)、肌肉发育(肌肉细胞大小和肌苷丝长度)和线粒体形态(循环和大小)的表型进行了定量分析,以了解基因突变对这些细胞结构的影响。这些定量方法并不限于此处描述的应用,因为它们可以很容易地用于定量评估线虫中其他组织和细胞器的形态,以及其他模型生物体中的形态。

C. elegans是研究不同组织和细胞器形态的理想模型有机体,由于其简单、已知的细胞系、透明度和可用的工具。在这里,我们提供定量方法,用于研究细胞器(例如线粒体)和组织,包括使用实时荧光成像和免费生物图像处理软件的突触和肌肉。
正常突触开发需要严格管理 MEC-17 表达
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Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

本研究概述了使用免费提供的图像处理工具对C. elegans的突触大小和定位、肌肉形态和线粒体形状的定量测量。这种方法允许将来在C.elegans的研究定量比较由于基因突变而导致的组织和细胞器结构变化的程度。

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

量化组织和细胞器形态变化的能力对于理解基因功能以及基因突变的影响非常重要。我们的协议解释了如何免费可用的软件可用于非主观地评估线虫（C.elegans）中突触、肌肉和线粒体的形态。许多以前的研究都依靠定性方法比较形态变化。然而，这些可能是有问题的，因为它们可能无法捕捉到微妙的表型差异，可能超过和低估变化，并主观评估。我们的定量方法为评估形态变化提供了更稳健、更不偏颇的方法。这些方法可以很容易地用于研究其他细胞结构或模型生物体的形态变化，以便定义基因功能和疾病相关突变的后果。首先准备幻灯片进行成像。将一滴融化的阿加罗斯放在显微镜幻灯片上，然后立即用另一张显微镜幻灯片向下按压液滴，轻轻压平糖。让加糖干燥一分钟，然后小心地将两个幻灯片分开，将凝固垫放在其中一张幻灯片上。将幻灯片放在显微镜台上，在 agarose 垫的中心添加 5 到 10 毫升的麻醉剂。使用热消毒的采摘，在溶液干干之前，快速将 10 到 15 只动物从库存板转移到麻醉液滴。然后应用盖玻片，将其定位在阿加罗斯垫的正上方，然后轻轻地放下。使用线扫描共声显微镜与 488 和 552 纳米光学泵送半导体二极管激光器耦合，并配备图像捕获软件，对突触区域进行成像。定位蠕虫后，切换到 40 倍放大倍率并应用浸入介质。通过 552 纳米激光器、1% 的 tagRFP 荧光磷功率和 488 纳米、2% 的 GFP 荧光量，通过刺激荧光的突触区域。使用混合光电探测器捕获图像，并设置增益，以防止荧光团过度暴露。根据目标，收集 Z 堆栈图像，使用最佳 Z 步法大小覆盖整个突触。要分析突触区域，首先将图像加载到 CellProfiler 3.1.5 软件中。设置 NameAndType 模块，并使用 UIS-115 转基因表示的漫反射 tagRFP 按手定义确定突触区域。通过将测量对象形状和测量对象突触模块添加到管道中，测量手动定义的突触的大小和荧光。然后通过添加计算Math模块，将从JSIS37中获得的集成强度单位除以从UIS-115获得的集成强度单位来计算相对集成的荧光强度。完成后导出所有测量和计算。要测量肌肉细胞区域，请打开斐济软件中的图像，并使用多边形选择来仔细跟踪单个倾斜肌肉细胞。通过拖动锚点来调整多边形的末尾的线，以改善跟踪。导航到软件顶部的"分析"选项卡，然后单击"测量"以计算所选区域。对于具有退化或缺失区域的肌肉细胞，使用多边形选择工具跟踪缺失区域，然后再次单击"测量"。如果有多个间隙，请分别跟踪每个间隙。计算间隙面积与整个单元格区域的比率。高比率表示肌肉退化的程度较高。如果没有缺失区域，则比率计算为零。打开弹性并在"创建新项目"下选择"像素分类"。然后重命名并保存项目。导航到输入数据选项卡，然后单击"添加新"，然后添加单独的图像。使用 TIF 文件将弹出窗口引导到文件夹，然后选择所需的图像。在"要素选择"选项卡中，单击"选择要素"以选择绿色勾选框指示的所有像素要素。此步骤中的像素选择用于区分下一步中不同的像素类别。使用训练面板来区分单个 GFP 表达肌素丝和不需要的背景。单击"添加标签"，在灯丝之间草草写不需要的背景和空格，开始分类器训练。添加第二个标签，将其重命名为"丝"，并在许多灯丝上乱写。单击"实时更新"以确保分类已正确完成。如有必要，添加更多草书以微调培训。执行训练分类器后，转到"预测导出"面板，然后单击"选择具有概率的导出图像设置"作为源。确保已勾选切口子区域 x 和 y，并且 c 已取消勾选。选择零和一作为 c 的开始和停止值，并将数据类型转换为未签名的 8 位。勾选重新规范化，然后将输出文件视频更改为 png，根据需要重命名文件和目录。关闭"导出设置"窗口并导出刚刚分段的图像。然后在斐济软件中打开 png 文件。使用默认设置调整图像的阈值并应用骨架化插件，这将创建单独的结果表。该协议已用于探索α-多布林乙酰转移酶 MEC-17在突触开发中的功能。对后/侧微管或PLM突触图像的定量分析表明，MEC-17的超表达会干扰正常的神经元功能。与野生型控制动物过度表达 MEC-17 相比，预突触区域和突触完整性显著降低。这些技术还用于分析夏科特-玛丽-牙2型或CMT2的C.elegans模型，该模型携带CMT2相关基因的突变。对身体壁肌形态的视觉评估显示，与野生类型控制相比，试验动物的缺陷增加了2.5至3.5倍。在fzo-1和uns-116中发生突变的动物经历了肌肉条纹损失、细胞碎片积累和肌肉纤维退化。虽然dyn-1突变的动物在肌肉形态方面的缺陷明显减少。fzo-1和uns-116突变体显示，与野生类型动物相比，差距与单个细胞总面积的比例增加了五到六倍，平均纤维长度也明显缩短。要对样本进行比较，必须确定图像采集的最佳条件，并确保这些条件得到一致使用。这些技术将允许研究人员用定量方法比较不同遗传背景的形态变化。这降低了主观性，因此有助于提高生成数据的可表示性。

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Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Unit 1

Cells, Genomes, and Evolution

SCIENTISTS IN ACTION

Measuring Caenorhabditis elegans Life Span on Solid Media

Measuring Caenorhabditis elegans Life Span on Solid Media

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

科研

生物学

A high-throughput method to globally study the organelle morphology in S. cerevisiae

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Analyzing and Building Nucleic Acid Structures with 3DNA

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...