Name: easy measurement of diffusion coefficients of egfp-tagged plasma membrane proteins using k-space image correlation spectroscopy
Uploaded: 2014-05-10T14:00:00
Description: Watch this Scientific Journal Video about Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy at JoVE.com

这项工作是由Lundbeck公司初级组组长奖学金到LNN支持。 PWW承认从加拿大自然科学和工程研究理事会（NSERC）补助资金支持。我们也感谢丹麦分子生物成像中心南丹麦大学访问旋转盘显微镜。

采集在质膜EGFP-标记的蛋白的KICS分析可以在落射荧光显微镜，TIRF设置或在一个旋转盘显微镜设置为获取所关注的膜的图像来完成。摄像机像素尺寸以及图像帧之间的时间是需要的分析。可用于分析在4-30赫兹帧频100-1,200帧的图像序列。在采集时，要保持对焦的膜在整个成像持续时间和集中于平膜，其中，荧光均匀分布的一小部分是很重要的。移动囊泡，突出膜区域和细胞器可以稍后在分析过程中?...

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本文介绍了如何应用KICS分析方法，从蛋白质标记荧光蛋白的显微镜图像确定扩散系数的详细一步一步的概述。该分析是独立探针的选择与标记密度非常广阔的动态范围，并且可以由此也可以应用到标记的量子点以及染料和荧光蛋白，例如绿色荧光蛋白的蛋白质。 计算该蛋白质的有效扩散系数，有几个关键步骤，这已经得到了优化。至关重要的是仅在膜图像标记的蛋白质。如?...

质膜蛋白质的四维时空组织和横向流动被严格调控，并可能在蛋白质的功能，活性和蛋白质 - 蛋白质相互作用中发挥作用。质膜蛋白的侧向扩散，在传统上被研究通过计算单个微粒的扩散系数由量子点的时间推移成像或染料标记的质膜蛋白2-4。这种方法需要插入质膜中的蛋白质对量子点或染料标记，它可以破坏蛋白质折叠和功能，因此，不能达到对某些蛋白质的胞外标记。量子点的空间体积已经显示出减缓蛋白5的扩散，而且，在人口中的蛋白质只有一小部分被标以量子点，并且如果该馏分是代表对总它不知道池质膜蛋白。单粒子TRA扩散系数从形象系列量子点标记的蛋白他妈的（SPT）的测量涉及的映射与二维高斯粒子的适应其次是广泛的分析算法成像的峰位。该分析在计算上是非常密集的，需要大量的非线性曲线拟合中的时间推移序列的每个图像帧的显微镜点扩散函数（通常为2维空间高斯）和粒子位置的后续联的近似成粒子轨迹描述单分子6,7的运动。 一项最近开发的图像相关技术，K空间图像相关光谱（KICS）使扩散系数的相对简单的测量的荧光标记质膜蛋白。经常性地计算由KICS标有荧光蛋白膜蛋白的扩散系数的可能性是它拥有独特的工具比传统的量子几个优点点SPT分析：外标签和费时外标签无插入是必需的（可用于表达荧光蛋白的细胞系）;扩散系数是从比较标记的量子点的子集的荧光蛋白的总萃取池;分析是简单而不需要跟踪单个蛋白质和分析，可以使用具有附加的用户编程不要求现有的代码来执行。该方法快速，因为它是一个平均的技术，它能够快速计算扩散系数和计算。这些快速扩散测量的蛋白质补充人口的艰苦SPT方法获得的总体的子集的更详细的分子运输信息。 KICS相关时间已先被转化为傅立叶空间荧光显微图像序列，从而分离FL由于从那些由于分子运输1光物理uorescence波动。其结果是，KICS可以确定数密度，流动速度和扩散的荧光而被中立通过复杂的光漂白或闪烁的荧光团标记的分子。这使得KICS用于快速确定荧光标记的细胞的膜蛋白的扩散动力学，而不需要自定义写入算法的有用工具。除了 ​​荧光标记的蛋白，KICS也可以应用到量子点标记的膜蛋白8。 本文提供了一步一步的介绍如何使用KICS演示了如何将作物，如何使用代码，以及如何评估产生的地块，提取绿色荧光蛋白标记的质膜蛋白质的扩散系数从中扩散系数进行萃取。作为一个例子，通过旋转圆盘镜组中半全内反射所获得的数据发荧光NCE（TIRF）模式标记EGFP肾水通道蛋白水通道蛋白3（AQP3）中，提出了。

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easy measurement of diffusion coefficients of egfp-tagged plasma membrane proteins using k-space image correlation spectroscopy

横向扩散和质膜蛋白的条块被严格调控的细胞，因此，研究这些过程，就会发现新的见解到质膜蛋白的功能和调节。最近，K空间图像相关光谱（KICS）1的开发，使扩散系数的常规测量直接从荧光标记细胞膜蛋白的图像，即避免探头光物理引入系统偏差。虽然用于分析的理论基础是复杂的，该方法可以通过使用一个免费提供的代码来测量蛋白质的扩散系数非专家来实现。 KICS计算时间相关函数从荧光显微图像堆栈的每个图像的傅立叶变换倒数（K-）空间后。随后，圆形平均，自然对数变换和线性拟合的相关函数产生的扩散系数。这本文提供了一步一步的指导，通过KICS图像分析和扩散系数的测量。 首先，使用荧光显微镜获取荧光标记的细胞质膜蛋白质的高帧频的图像序列。然后，感兴趣区域（ROI）避免细胞内细胞器，移动囊泡或凸膜区域的区域被选中。投资回报率堆栈被导入到一​​个可以自由使用的代码和几个定义的参数（见方法部分）是为KICS分析设置。然后程序会生成一个情节从k空间时间相关函数“的坡坡”，而扩散系数是从该曲线的斜率计算。下面是一步一步的KICS程序使用肾水通道蛋白-3标有绿色荧光蛋白作为一个典型的例子来衡量一个膜蛋白的扩散系数。

为了获得合适的图像栈与KICS进行分析，不同的显微镜系统都可以使用。因此能够分析来自一个落射荧光显微镜，以及一个TIRF或旋转盘设置图像序列。该膜必须是平的，没有任何大的运动细胞器或移动囊泡/对象。本文提出AQP3的具有标记的EGFP在成象在一个旋转盘显微镜的焦点设定到质膜在9.2赫兹帧频活MDCK细胞一缩时图像序列。的焦点被设置在基底（底）质膜。该数据已出版的AJP细胞13。 </...

Watch this Scientific Journal Video about Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy at JoVE.com

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

本文提供了一个一步一步引导到波动分析技术K空间图像相关光谱（KICS），用于测量扩散系数的荧光在活的哺乳动物细胞标记质膜蛋白。

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 ...

Research

JoVE Journal

Biology

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

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) ...

科研

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Crystallization of Membrane Proteins in Lipidic Mesophases

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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

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

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...