谁最先开发这项技术在Sive实验室的博士詹妮弗Gutzman许多感谢。达纳 - 法伯癌症研究所的波士​​顿，马萨诸塞，慷慨地提供CAAX - GFP基因的质粒编码也感谢JB绿色。白石WM凯克基金会生物成像设施进行共焦成像。 HS是由NIHMH70926支持。 EG是支持由美国国家科学基金会博士后奖学金。

1。准备注射基因<ol><li>在此过程中使用的mRNA是由质粒编码CAAX - EGFP（memGFP）mRNA的转录。首先线性化的质粒，根据2008年等，Gutzman。 </li><li>然后抄写memGFP mRNA的使用mMessage mMachine套件。 </li><li>稀1μg/μl，导致mRNA的等份，并储存在-80 ° C。 </li><li>在一个细胞阶段的胚胎车道​​的宽度，准备1％琼脂糖的注塑模具。 </li><li>在注射前一天，成立了分离的女性，男性的交配笼。 </li><li>注射日，拉毛细管，使用微量车夫准备注射针。 </li></ol> 2。注射的mRNA <ol><li>在注射日，解冻1μg/μlmemGFP的mRNA在冰上的等份。 </li><li>在水稀释的mRNA 1:5的200ng/μl最终浓度，并保持在冰上。 </li><li>负载200ng/μl准备memGFP表达加入1μl毛细管针。 </li><li>装入一个天然气为动力的微量的一个显微针。 </li><li>调整的注射量1nl。 </li><li>野生鱼交配笼前一天拉的分隔。 </li><li>收集胚胎，尽快为他们规定。东方他们就在身边离的显微单细胞胚胎培养基（韦斯特，1995年）所覆盖的琼脂糖注塑模具。 </li><li>注入通过蛋壳和蛋黄等，直接存入单细胞的mRNA。注入约50％的胚胎实验。不要注入的细胞分为。 </li><li> 28 ° C过夜胚胎培养基孵育胚胎</li></ol> 3。安装和成像胚胎<ol><li>挂载和形象的胚胎，删除感兴趣的时间点之前的钳体视显微镜下一个一小时的胚胎绒毛膜。 </li><li>准备安装多达4胚胎幻灯片。 <ul><li>使用有机硅真空润滑脂密封盖玻片上的一个特别设计的一个洞，大约直径1cm 2mm厚的塑料滑动的底面。 </li><li> 0.7％琼​​脂糖在幻灯片的填充孔，静置约20分钟或直到硬化</li><li>一旦琼脂糖凝固后，取出一个小插件，使用200μL移液器形成圆柱形的孔，在安装每个胚胎。 </li></ul></li><li>广场上的琼脂糖凝胶填充的幻灯片，在解剖显微镜下的胚胎。添加tricaine50μL（韦斯特，1995年）麻醉胚胎。 </li><li>使用镊子对基础盖玻片利益与大脑或地区在琼脂糖圆柱孔定位的胚胎。 </li><li>硅真空润滑脂保护另一盖玻片盖在琼脂糖胚胎。 </li><li>图片胚胎使用一个倒置，荧光，激光扫描或旋转盘共聚焦显微镜。在63X或更高收集单个细胞内的神经上皮的高清晰度图像的图像。 </li><li>图像TIFF文件从LSM软件出口和使用Photoshop进行分析。 </li></ol> 4。代表性的成果/成果在图1所示是24 HPF斑马鱼神经上皮中脑和后脑心室之间有代表性的共聚焦图像。每个单元是与膜结合的GFP标记。 <img src="/files/ftp_upload/1217/Graeden JoVE figure 1.png" alt="" width="450" height="449" /> 图1。现场共焦成像一个memGFP 24 HPF注入斑马鱼的胚胎。 （一）神经上皮与GFP的概述的每个细胞的胚胎24 HPF。成像区分开的中脑和后脑脑室（M，H），形成了中脑，后脑边界。

<ol>
	<li>Gutzman, J. H., Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech Dev. 125 (974), 11-12 (2008).</li><li>Westerfield, 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> The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish. , (1995).</li></ol>

在这段视频中，我们表现出的mRNA注射到单细胞的斑马鱼胚胎的方法。在这里，我们使用一种膜定位GFP标记每个细胞的基因编码。然后，我们演示了如何安装和形象在单细胞分辨率的发育中的大脑。这项技术使我们能够研究一种新型的细胞形态变化，基部收缩，形成中脑，后脑边界（Gutzman等，2008）所需。类似的分析等现象有可能显着扩大我们对脊椎动物的形态发生和底层细胞生物学的理解。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>SeaKem GTG agarose</td>
<td> Reagent</td>
<td>Lonza</td>
<td>50027</td>
<td>&nbsp;</td>
<tr>
<td>3-aminobenzoic acid ethyl ester (Tricaine)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>Tool</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>Borosil 1.0mmOD x 0.5mm ID/fiber 100mm</td>
</tr>
<tr>
<td>Silicone vacuum grease</td>
<td>Reagent</td>
<td>VWR</td>
<td>W0S717</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11232-20</td>
<td>Dumont #5 Bio Inox</td>
</tr>
<tr>
<td>1-200 &#x03BC;l Pipette tips</td>
<td>Tool</td>
<td>Tip One, US Scientific</td>
<td>111-0806</td>
<td>These are the correct size for 24 hpf embryos. </td>
</tr>
<tr>
<td>mMessage mMachine transcription kit</td>
<td>Reagent</td>
<td>Ambion</td>
<td>AM1340</td>
<td>SP6 RNA polymerase</td>
</tr>
<tr>
<td>Zeiss LSM 510 scanning confocal </td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Micromanipulator</td>
<td>Tool</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Microinjector</td>
<td>Tool</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PLI-100</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>Tool</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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live imaging of the zebrafish embryonic brain by confocal microscopy

在这段视频中，我们展示了我们的实验室已发展到分析细胞形态的变化和需要弯曲和折叠的发展斑马鱼脑（Gutzman等，2008）的重排的方法。这种分析提供了一次脊椎动物大脑的三维结构的发展所需的基本细胞生物学的新认识，并显着提高我们的能力，以研究神经管的形态发生。胚胎斑马鱼的大脑形脑室神经上皮内膨胀的开端，在18小时后受精（HPF）。 24 HPF，神经管的形态发生的最初步骤，是完整的。使用这里介绍的方法，在细胞阶段的胚胎基因编码的膜，有针对性的绿色荧光蛋白（memGFP）注射。后注射和孵化，胚胎，目前18至24 HPF，安装，倒置，在琼脂糖和激光共聚焦显微镜成像。值得注意的是，斑马鱼的胚胎是透明的，使其成为理想的荧光成像系统。虽然我们的分析集中在中脑，后脑边界和后脑，这种方法可以延长任何地区的分析，在深度为80-100微米的斑马鱼。

Watch this Scientific Journal Video about Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy at JoVE.com

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

在这段视频中，我们表现出的方​​法，通过它来分析生活在单细胞共聚焦显微镜分辨率的斑马鱼胚胎发育中的脊椎动物大脑。这包括我们注入单细胞的斑马鱼胚胎的方法，通过它，随后装载和图像的大脑发育。

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

live-imaging-of-the-zebrafish-embryonic-brain-by-confocal-microscopy

Nanyang Technological University

Research

JoVE Journal

Biology

18.5K 次观看.  MIT - Massachusetts Institute of Technology. 在这段视频中，我们表现出的方​​法，通过它来分析生活在单细胞共聚焦显微镜分辨率的斑马鱼胚胎发育中的脊椎动物大脑。这包括我们注入单细胞的斑马鱼胚胎的方法，通过它，随后装载和图像的大脑发育。

视频: Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...