作者想感谢他的意见和埃克塞特大学的生物成像设施的埃克塞特大学教授尼古拉斯米尔诺夫。经费是由英国生物技术和生物科学研究理事会（赠款参考BB/E002682/1）。

下面给出的协议描述了一个简单的方法，使用PFD的浸润安装在拟南芥叶片中等，但我们预计，这种方法可用于成像空气丰富的组织需要的各种应用。 1。安装在PFD的叶样品<ol><li>准备透气垫片的二甲基硅氧烷（PDMS，卡罗来纳州观察凝胶）载玻片。硅橡胶是一种粘弹性聚合物，可以被塑造提供符合实验要求一腔。 </li><li>与空气平衡PFD的。这可能是实现与空气气泡或晃动PFD功能，在一个充满空气的瓶子体积小。 </li><li>倒出PFD的一个开放的培养皿中，并飘起了一个全苗或PFD的叶切除5分钟。该组织应成为半透明，让人联想到玻化组织（图2（a） ）。叶可能会出现比之前曝光变暗或更轻PFD的，依赖丁照明条件和年龄组织使用。 </li><li>与空气平衡的PFD填充PDMS室，仔细从孵化培养皿转移的PDMS厅组织样本。盖玻片和形象，根据实验要求密封的幻灯片。 </li><li>注：PFD的也表现出良好的盖玻片上构建了一个开放室或在灌注室和兼容硅脂。 PFD的聚四氟乙烯组件不兼容，因为它溶解。 </li></ol> 2。代表性的成果： PFD孵育叶标本的显微镜检查显示，大部分空域充斥。图2（b）显示在PFD的暂停重组的GFP淹没的空域。很明显，叶与PFD的潜伏期后淹没，但偶尔口袋里的空气可能会保持。水不洪水在这些条件下的叶。培养和安装在样品的激光扫描共聚焦显微镜<html>红外，水或PFD显示，PFD的给出了一个对水和空气的优势可能在成像深度（ 图3）。在图3给出的例子表明，空气，水和PFD 安装拟南芥叶表达cytosolically本地化金星，表示35S启动子下组成了 11 YFP的变种。比较PFD和水时，我们可以看到成像深度约2倍。但是，应该指出，使用PFD时看到精确的改善与数值孔径的镜头和使用的组织类型不同。我们已经看到细胞质流PFD的治疗苗发根伸长，每个健康植物的指示。此外，我们有2所示，Fv / Fm的 ，12植物光合运作的措施仍然在成像使用的时间表，容忍的限度内。 3394fig1.jpg“/&gt; 图1。植物叶的复杂性和光的影响 图解代表性的A.解剖特点拟南芥叶光学设置。 OBJ的缩写。 =物镜，入境事务处处长。 =浸入液体，覆盖。 =盖玻片，产妇和新生儿破伤风。 = mountant，切。 =角质层，广告。 EP。 =近轴面表皮，ST。 =气孔孔径，SP。 =海绵叶肉，=空气空间，PAL。 =栅栏叶肉，VB =维管束，广告。 EP。 =近轴表皮。细胞壁是由黑线表示。 <img alt="图2。" src="/files/ftp_upload/3394/3394fig2.jpg" /> 图2。 PFD的容易渗透拟南芥叶 （一）培养叶片在空气，水或5分钟PFD的成像徕卡DCF3000FX数码相机使用徕卡MZ16F显微镜（莱卡微系统（英国）有限公司，米尔顿凯恩斯，英国）。朱庇特的标志印在醋酸纤维膜和照明来回米以下的灯箱。曝光时间为89毫秒，所有图像采集和处理相同。标尺代表2毫米。 （二）携带PFD的纯化的重组绿色荧光蛋白（GFP）分隔体内的空域。 GFP是假的绿色，红色是用来显示自体荧光，分隔叶肉细胞叶绿素。 （ 图2（b），权限和Littlejohn等提供全面的技术细节再现。2010年2 ）。标尺代表25米。 <img alt="图3。" src="/files/ftp_upload/3394/3394fig3.jpg" /> 图3。 PFD的提高叶片中的共焦成像 激光共聚焦显微镜图像显示cytoplasmically本地化金星荧光（绿色）和叶绿素的自发荧光（红色）在完整的拟南芥叶片在空气，水或PFD成像。激发波长为514 nm和荧光光谱在518纳米金星为604纳米和657纳米679纳米为叶绿体。每个小组是一个单一的焦节从11每隔5微米收购的激光共聚焦图像的Z - Stack。分别提取59影像在1微米的间隔，并已用于生产补充电影获得一个完整的Z - Stack的。深度测量相对于表皮的表面。代表图像，这是相同的处理，显示。实验细节，如显微镜设置在Littlejohn等人 ，2010年2发现的相同。 Scalebars代表20微米。

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, 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+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <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+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

没有利益冲突的声明。

这是一个简单和易于使用的技术，以提高显微镜充满空气或光学复杂的组织。我们已经表明，该技术有一些具有很强的优势，我们希望这将是用于澄清有关空气丰富组织的生物学问题。例如，这将是一个研究植物叶肉或肺部的病原体攻击的自然选择。我们也意识到该技术的局限性。我们更好的折射率匹配工作，使用其他交通工具显微镜，并在较长的时间尺度实验的全氟化碳mountant增资。我们还认识到，虽然PFCs的主要优点之一，即生物惰性有一个不利的一面。 PFCs的不容易溶解的生物分子，这也意味着，他们不能很容易地用来提供利益，如激素，药物和其他小分子或离子的化合物。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>全氟萘烷</td><td> F2化学品</td><td> N / A </td><td>电话订购。 </td></tr><tr><td>卡罗来纳州观察凝胶</td><td>叶片生物</td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

到生物样品中获取高分辨率图像深的问题是普遍承认 1。在充满空气，如海绵状植物叶片或脊椎动物肺部进一步的困难，出现多个过渡之间的细胞和生物组织的光学系统的其余部分之间的空域和细胞成分，折射率叶肉组织。此外，折射率不匹配导致荧光激发和荧光显微镜所排放的信号衰减。我们在这里描述的应用程序的全氟化碳，全氟萘烷（PFD），浸润成像介质光学提高激光扫描共聚焦显微镜（LSCM）样品成像深度，而不采取激光功率的损害增加，和最小的生理影响2。我们描述了PFD的使用拟南芥叶组织，这是光作为其结果的复杂的协议结构（ 图1）。 PFD的有属性，使之适合使用本3。 PFD的折射率（1.313）与水（1.333）相比和细胞质（约1.4），比空气（1.000）。此外，PFD的是现成的，无荧光，是无毒的。 PFD的低表面张力（19达因厘米-1）低于水（72达因厘米-1），也低于气孔渗透，这使得它洪水的限制（25 - 30达因厘米-1）海绵叶肉空域没有一个潜在的破坏性的真空或表面活性剂的应用。最后，至关重要的是，PFD的大容量为溶解的CO 2和O 2，它允许气体交换，以保持在洪水淹没的组织，从而最大限度地减少对样本的生理影响。这些属性已被用于各种应用，其中包括部分液体呼吸和肺inflaTION 5,6，7手术，人造血液，增长充氧媒体9，和冰晶体的形成在植物10研究。目前，它是常见的，安装在现场共焦成像的水或缓冲溶液的组织。我们认为，作为安装介质的使用PFD的代表对改善现行的做法，使整个成像活叶样品制备简单。

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

我们形容一个浸润安装介质使用的全氟萘烷。这是一个简单的方法，为提高成像深度<em&gt;拟南芥</em&gt;叶组织以最小的生理影响。

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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Research

JoVE Journal

Biology

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K 次观看.  The University of Exeter. 我们形容一个浸润安装介质使用的全氟萘烷。这是一个简单的方法，为提高成像深度拟南芥叶组织以最小的生理影响。

视频: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

使用定量成像技术鉴定和表征异源细胞群体

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...