Name: measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching
Uploaded: 2010-02-26T16:45:00
Description: Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

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...

<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) ...

Research

JoVE Journal

Biology

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

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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

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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

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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

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

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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)

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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 ...

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

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

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

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

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Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...