Name: sample drift correction following 4d confocal time-lapse imaging
Uploaded: 2014-04-12T22:35:00
Description: Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

我们要感谢盖比马丁斯和EMBO2010 3D发展成像的研讨会，这项工作开始，所有的贡献者斐济和ImageJ的项目的组织者。

1，4D时间推移成像实验用于图像采集的设置会有所不同取决于所使用的设备上。共焦显微镜进行光学部分的样品的能力取决于多个因素：激励，针孔大小，客观的数值孔径，样品的折射率，并且其中样本被嵌入该介质的波长。共焦针孔选定的大小将决定所收集的光学部分的厚度。一个更小的针孔会产生更薄的光学部分增加了Z轴的分辨率，但降低了光的捕获量。一个更大的针孔...

<ol>
	<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>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 42-42 (2004).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+Microscopy.">ImageJ for Microscopy.</a> BioTechniques. 43 (S1), 25-30 (2007).</li><li>Linkert, 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE transactions on image processing : a publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Kuglin, C. D., Hines, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+correlation+image+alignment+method.">The phase correlation image alignment method.</a> Proceedings of the IEEE, International Conference on Cybernetics and Society. , 163-165 (1975).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+motility+and+actin+cytoskeleton+of+individual+neurons+and+neural+crest+cells+in+zebrafish+embryos.">Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.</a> J VIs. Exp. (36), (2010).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+extrusion+from+the+epidermis+of+developing+zebrafish.">Live imaging of cell extrusion from the epidermis of developing zebrafish.</a> Journal of Visualized Experiments: JoVE. (52), (2011).</li><li>Benard, E. L., vander Sar, A. M., Ellett, F., Lieschke, G. J., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments: JoVE. (61), (2012).</li><li>Nguyen-Chi, M. E., 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=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

我们使用后处理软件来纠正从延长的时间推移显微镜实验得出的数据集的样本漂移的能力受到许多因素的限制。辨别漂移对样品的迁徙运动能力依赖于所使用的细胞标记。无论是否广泛的样本中明示或图像采集过程中不涉及迁徙事件细胞标记为漂移校正的最佳来源。该插件使用单个信道来注册的时间点之间的移动，然后应用这一注册到所有收集到的图像的信道。因此，可能有利的是使用两种荧光?...

共焦成像被广泛应用于细胞和发育生物学跟随细胞运动和变化的形态。拍摄的一系列光学切片在不同的焦平面允许生成一个三维（3D）模型的样品上，然后可以通过创建时间推移一系列的三维数据集被扩展成四个维度（4D）的。 4D数据集的产生使细胞运动和行为的详细的测量。在长期的延时实验是很常见的观察样品的运动。这可以通过在硬件控制阶段和焦点位置轻微的不准确引起的。而在另一些情况下，漂移是诱导样品安装介质内的样品生长或弹性运动的结果。方法存在补偿或限制这些运动包括改善硬件聚焦系统和安装介质的刚性增加。然而，这些方法不能在许多情况下应用，由于摄像s等最多需要提供适当的条件下对样品维持和生长。开源软件解决方案确实存在运动的二维校正随着时间的推移，通过使用STACKREG和TurboReg（http://bigwww.epfl.ch/thevenaz/stackreg/）5插件在ImageJ的或斐济，但这些都不能被应用到4D数据集。 为了校正样品的漂移，我们已经开发了一个插件（正确的3D漂移）利用开源的图像处理平台，斐济1。我们的插件能够执行相位相关配准修正的发生是由于在三维时间推移实验样品的漂移而导致的运动。相位相关6是计算效率的方法来确定图像之间的转换。这里所描述的插件，利用由Preibisch 等 7开发了相位相关的库。在多通道的实验中，该插件利用一个信道到determiNE所需的校正。这种校正，然后应用到产生的4D数据集的登记任何额外的通道。 在斑马鱼的模型系统，可以进行延时成像一段许多小时，甚至数天8。用于安装在斑马鱼的常用方法是嵌入麻醉活胚胎在低熔点琼脂糖（0.8-1.5％），限制其运动9-11。而移动被限制在样品的生长仍然发生，导致换档位置的视场范围内的单元格。为了遵循这个胚胎内细胞的运动，有必要首先纠正为整个样本的移动。这个协议是与斑马鱼标本研制，并已用于图像体节发育12而是可以应用到任何4D共聚焦数据集。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

的四维（4D）的共聚焦数据集的产生;由3D图像序列的一段时间内;提供了一个极好的方法来捕捉参与发育过程的细胞行为。跟踪并按照细胞运动的能力是由发生是由于图像采集过程中的漂移的样品，或在一些情况下，生长的样品的运动的限制。跟踪细胞集受漂移和/或生长将把这些运动到细胞位置的任何分析。这可能会导致在样品内的静态结构的表观运动。因此，之前小区的跟踪，任何样品漂移应予以纠正。使用ImageJ的2,3开源斐济分布1及注册成立LOCI工具4，我们制定了正确的3D漂移插件的共聚焦数据集删除错误的样运动。该协议有效地补偿了样品翻译或改建焦宝sition通过利用相位相关注册的每个时间点的一个四维数据集焦，同时保持可视化和测量细胞运动在延长的时间推移实验的能力。

在发展中的斑马鱼，快肌细胞融合成多核纤维由19小时后受精（20 -体节期）13。为了可视化核的运动和融合肌细胞，我们进行了使用转基因品系的骨骼α-肌动蛋白启动子的控制下表达绿色荧光蛋白（GFP）标记所有肌肉的4D共聚焦延时成像的细胞14和注射编码红色荧光蛋白的mCherry标记的核定位序列，以标记细胞核RNA。注入的转基因胚胎被安装在低熔点琼脂糖和图像序列组成的在2.0微?...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

样品漂移校正继4D共焦时间推移成像

时间推移显微镜可发育过程的可视化。图像采集过程中的增长或样品的漂移减小到准确跟踪和开发过程中测量细胞运动的能力。我们描述了使用开源的图像处理软件，以纠正随着时间的推移三维样品漂移。

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

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