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。一个更大的针孔会增加光学部的厚度，从而降低沿z轴的分辨率，但增加光捕获量。 其他因素集合的4D数据之前修正过程中考虑的因素包括: <ol><li>优化扫描速度，删除或限制，漂移ðuring一个时间点的捕捉。因此，采取的图像采集时间应的时间点之间的时间间隔的一小部分。 </li><li>完全重复结构或网格，不适合作为结构注册为不可能确定所需的校正。随机分布的珠或不均匀结构将使明确登记。 </li><li>如果漂移是意料之中的，增加扫描区域和上下聚焦限度，以确保感兴趣区域仍然是图像堆栈内。 </li><li>除了确保有适当的空间分辨率来解决利益结构，设置采样率的动态事件正在研究提供时间分辨率。 注:因此图像的时间点之间的时间应定期重复事件之间至少有一半的时间间隔，减少不规则的事件。 </li></ol> 2，打开激光共聚焦ðataset 开源包斐济是ImageJ的程序，其中包含预先安装的插件从显微镜实验收集到的数据进行大量的流程的分布。该软件提供更方便的插件更新架构，包括正确的3D漂移插件用于该协议的副本。该软件支持的专有显微镜图像格式繁多的通过使用开放式显微镜环境的生物的格式导入插件导入。 <ol><li>安装斐济程序（http://fiji.sc/Downloads）。 </li><li>使用LOCI生物格式进口商插件加载收购数据集。 </li><li>如果数据集是不是分配给程序的内​​存较大，选择内存管理部分中的"使用虚拟堆栈选项"。 </li></ol> 3，修正了3D对象的漂移在后处理在延长的时间推移的过程中实验嵌入式即使样品可能会移动。以纠正任何运动，并允许成像的迁移事件进行分析，能够进行图像的后处理。所有图像后处理必须在这项工作产生的任何分析的方法加以明确说明。 <ol><li>一旦数据集加载，运行正确的3D漂移插件。 </li><li>如果有多个图像通道，选择要用于注册的图像的通道。这应该理想地代表了样品中的静态结构，而不是任何迁徙或移动元素。但是，如果这是不可能用最少的运动通道应选择。 </li><li>如果用于该分析系统上的可用RAM是原始数据集小于两倍的大小，选择使用虚拟堆栈选项。这将在注册hyperstack存储为图像序列，而不是保存到RAM中的文件。 <ol><li>选择一个文件夹的插件输出单个纠正IMAGES文件。一个单独的图像文件将每个通道在每个z位置创建。 </li></ol></li><li>该插件会再进行每个时间点之间的成对相位相关分析，以确定所需的校正它，然后应用到数据集。 </li></ol>

<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>

作者宣称，他们有没有竞争的财务权益。

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

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。

样品漂移校正继4D共焦时间推移成像

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

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K 次观看.  Monash University. 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。 

视频: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...