这项工作是由一个部国防时代的希望学者奖（W81XWH - 05 - 0396号）和皮尤学者在生物医学科学奖，以爱德华B.布朗第三。

1。校正光学系统。 MP - FRAP实验的关键设备包括：一个锁模激光源，电光细胞（束调制），脉冲发生器，分色，物镜，荧光过滤器，门控光电倍增管，和数据记录系统（光子计数器和多通道定标器）。 2。确定安全监察权力。 <ol><li>激光衰减低，但合理的，样品中的荧光产生的力量。 </li><li>聚焦在样品</li><li>设置一个光子计数器集成在一个时间尺度更长的时间比预期的荧光恢复。随着焦距的体积举行的样本内固定（这是由一个点扫描来实现我们的显微镜软件），记录一个光子计数数据的合理数量的。 </li><li>增加动力，并采取一系列的另一个光子计数数据。重复，直到在光子计数的增加，这似乎与尊重的权力增加显着减少。 </li><li>剧情日志（光子计数）作为日志（电源）的功能。从两个斜坡的偏差显示在监视器的漂白。选择低于此切断电源，显示器的电源，但它允许一个合理的信噪比。 </li></ol> 3。确定输入参数的脉冲发生器和多通道定标器。 <ol><li>决定备份的信封根据文献和/或斯托克斯 - 爱因斯坦方程和扩散方程的扩散系数和恢复时间的估计。 </li><li>设置必要的参数值上的脉冲发生器和倍线器。 <ol class="lalpha"><li>脉冲发生器，设置预漂白和漂白剂次。一个拇指规则是，预漂白剂应比预期的一半恢复时间的1-2倍，漂白时间应十分之一的预期的一半恢复时间。 </li><li>洁牙机，设置斌宽度约一个半漂白工期，并设置每个记录箱的数量，这样的产品是您选定的bin宽度和每个记录箱大于或约等于你的预期的全面复苏，加上前期漂白剂和漂白剂次。 </li><li>返回的脉冲发生器，并设置pre-bleach/bleach/monitor序列的频率等于一个值仅比的bin宽度倍的每个记录箱产品的倒数较小。 * </li><li>最后，设置的记录数/扫描定标器，在您所选择的显示器的电源信号强度的基础上。 *（1/frequency）脉冲发生器上设置一个完整的pre-bleach/bleach/monitor序列的时间比数据采集时间的洁牙机（斌宽x箱/记录），这是非常重要的。如果不符合这个标准，可能会出现多个漂白触发内的洁牙机上的单个记录。 </li></ol></li><li>显示器的电源设置到可接受的水平，确定以前。将漂白功率200兆瓦或以上显示器漂白试验期间确定的漂白截止。这些权力都是不同的电压设置跨PC水晶。 </li><li>密封显微镜，关掉灯，打开光电倍增管，开始点扫描显微镜软件，然后启动脉冲发生器和洁牙机。 </li><li>恢复曲线绘制曲线和标记三个要点：1）预漂白荧光，2）荧光立即漂白后，和3）在数据集的结束荧光分析。使用荧光值前，立即后的漂白剂，以更好地估​​计半恢复时间。 </li><li>有了这个半恢复时间的估计，使用的经验法则前面提到预漂白剂，漂白剂，以确定新的价值观，和bin宽度工期。此外，使用数据集结束前的漂白剂荧光和荧光，以更好地估​​计实现全面复苏所需的时间。作为一个经验法则，应收集数据为一个时间段，允许的全面复苏，下半年的数据报道。 </li><li>调整的脉冲发生器和倍线器的参数，一个新的曲线，并继续调整参数，直到你的曲线呈现出强烈的漂白剂和顺利回收。漂白时间可以记住，并应减少，下面的经验法则，如果可能的话。漂白的权力也可能被调整，如果过浅，或过深，漂白剂，是实现。 </li></ol> 4。测试励磁饱和。 <ol><li>使用适当的监测和漂白剂的权力，确定以前，采取了一系列MP - FRAP曲线。每个恢复，规范化的预漂白荧光分析，使用适当的数学形式和非线性最小二乘拟合算法（见“数据分析”一节）。记录的漂白​​剂的深度参数的拟合值。</li><li>继续采取和分析漂白电源增加值连续系列MP - FRAP曲线。 </li><li>剧情日志（漂白深度参数）日志（电源）功能。从两个斜坡的任何偏差表示励磁饱和。选择漂白力量，产生了强烈的漂白剂，但不会导致饱和。 </li></ol> 5。继续采取MP - FRAP曲线。 <ol><li>继续采取曲线，上面一样，将所有参数设置正确。 </li></ol> 6。数据分析。 <ol><li>从荧光数据删除的预漂白和漂白剂的数据，设置和调整的时间等数据，T = 0对应立即漂白后的荧光值。 </li><li>规范化方面的平均预漂白荧光产生的荧光恢复。 </li><li>使用适当的数学模型和一个非线性最小二乘拟合算法，如在MATLAB中的lsqcurvefit功能，适合标准化曲线。这里提出的模型帐户扩散和对流的影响荧光回收率： <img src="/files/ftp_upload/1636/1636eq.jpg" alt="方程" /> F O漂白前的平均荧光，β是漂白剂的深度参数，τD为特征的扩散恢复时间，R为径向（W R）的宽度的轴向平方米的比例 （W Z）双光子焦距量，τVX = W R / V X，τVY = W ř / V Y，τVZ = W Z / V Z，V 2 = V + V Y 2 × 2 + V Z 2是对流的速度。对于密闭的成像平面的流量，τVZ→∞和1 /τVX + 1 /τVY可以换成1 /τVR 。在对流的情况下，分子中的两个指数函数为1，表达的是大大减少，只有两个拟合参数，β 和 τ剐。扩散系数D =瓦特R 2 /8τð很容易计算。 </li></ol> 7。代表性的成果。 <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="图1" /> 图1。代表性的漂白剂和恢复FITC - BSA的曲线。恢复良好的曲线呈现出较强的漂白剂和平稳回升，报告为下半年的数据完全恢复的荧光。 <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="图2" /> 图2。FITC - BSA的规范化复苏曲线（红色）和相关的最小二乘（黑色）。这适合产量的52.9 UM2 / S的扩散系数，与文献报道一致。

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li><li>Sullivan, K. D., Sipprell, W. H. B. r. o. w. n., Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo.">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li></ol>

多光子荧光漂白后恢复的力量在于，它能够探测与三维决议厚的样品。由于其在1990年的发展，MP - FRAP已被用来确定在细胞体， 体外厚的组织切片，并在体内组织和间质的扩散系数（或类似的交通参数） 。在这篇文章中，我们提出了必要的设备运行MP - FRAP实验，以及调整光路中的正当程序，设置实验参数，数据，以及恢复曲线分析。 适当的激发波长和发?...

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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

多荧光漂白后的恢复是一个显微技术用于测量大分子物质的扩散系数（或类似的交通参数），可同时适用于<em&gt;在体外</em&gt;<em&gt;在体内</em&gt;生物系统。多荧光漂白后的恢复是由漂白利益的地区在激烈的激光闪光荧光灯使用的样品，然后衰减梁和监控以外的利益弥漫的地区仍然荧光分子的荧光来代替光漂白分子。我们将开始通过普克尔盒（激光调制器），通过激光扫描盒样品和物镜光路沿激光束对准我们的演示。为方便起见，我们将使用荧光染料水溶液的样品。然后，我们将确定适当的实验参数为我们的样本，包括监视和漂白权力，漂白时间，斌宽度（光子计数），和荧光的恢复时间。下一步，我们将采取恢复曲线，很大程度上可以通过LabVIEW的（美国国家仪器公司，美国德克萨斯州奥斯汀​​）自动化，提高吞吐量的过程描述的程序。最后，扩散系数是由装修恢复数据到相应的数学模型，采用最小二乘拟合算法，可随时通过软件，如MATLAB（MathWorks的Natic​​k市，马）。

Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

在这篇文章中，我们将描述测量扩散系数，使用多光子荧光漂白后恢复的过程。我们将开始通过调整样品沿光路的激光，并确定适当的实验参数，然后继续产生，最后装修荧光恢复曲线。

通过双光子荧光恢复漂白后测量扩散系数

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

measuring-diffusion-coefficients-via-two-photon-fluorescence-recovery

Research

JoVE Journal

Biology

11.1K 次观看.  University of Rochester. 在这篇文章中，我们将描述测量扩散系数，使用多光子荧光漂白后恢复的过程。我们将开始通过调整样品沿光路的激光，并确定适当的实验参数，然后继续产生，最后装修荧光恢复曲线。

视频: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

通过双光子荧光恢复漂白后测量扩散系数

摘要

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此视频中的章节

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

有丝分裂的siRNA转染后的时间推移成像

在现场的斑马鱼胚胎的基于双光子Photoactivation

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

测绘在细胞膜分子扩散，多目标跟踪（MTT）

精子冷冻保存和恢复使用I ·冷冻包

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

恢复成年斑马鱼的心脏的高通量应用

细胞内钙的双光子成像

通过双光子荧光恢复漂白后测量扩散系数

摘要

探索更多视频

此视频中的章节

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

有丝分裂的siRNA转染后的时间推移成像

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

测绘在细胞膜分子扩散，多目标跟踪（MTT）

精子冷冻保存和恢复使用I ·冷冻包

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

恢复成年斑马鱼的心脏的高通量应用

细胞内钙的双光子成像

在现场的斑马鱼胚胎的基于双光子Photoactivation