Data Analysis for Total Internal Reflection Fluorescence Microscopy.
Data Analysis for Total Internal Reflection Fluorescence Microscopy.
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DOI：
10.1101/pdb.prot085571
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发表时间：
2016-05-02
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通讯作者：
Asbury CL
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文献类型：
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作者：
Asbury CL
在我们用来分析全内反射荧光（TIRF）的显微镜中，使用二向色滤光片将发射的荧光彩色地分裂成两种或三种不同的颜色（“通道”）。在我们的双色仪器中，将绿色发射波长（405-488 nm;用于成像绿色荧光蛋白[GFP]标记的蛋白质）和远红发射波长（650-800 nm;用于成像Alexa-647标记的微管）分别投射到单个相机的上半部分和下半部分。可以交换单个滤光片以收集近红波长（561-640 nm;用于成像mCherry或Alexa-568标记的微管）而不是远红。我们的三色仪器非常相似，除了绿色、近红色和远红色范围被投射到三个独立的相机上。在任何一种情况下，不同的颜色都可以同时成像。通常情况下，我们以10帧/秒的速度收集图像，持续约200秒。我们已经开发了一系列的半自动图像分析程序，在LabView中编写，以获得亮度，停留时间，和单个颗粒的流动性绑定到单个微管。基本的分析步骤很简单，也可以使用ImageJ或Matlab来实现。为方便起见，该方案描述了单个微管的分析。需要来自许多实验性试验的许多微管的数据来获得独立于随机和试验间变异性的可靠结论。
In the microscopes we use to analyze total internal reflection fluorescence (TIRF), the emitted fluorescence is split chromatically, using dichroic filters, into either two or three different colors (“channels”). In our two-color instrument, the green emission wavelengths (405–488 nm; for imaging green fluorescent protein [GFP]-tagged proteins) and far-red emission wavelengths (650–800 nm; for imaging Alexa-647-labeled microtubules) are projected onto the upper and lower halves, respectively, of a single camera. A single filter can be swapped to collect near-red wavelengths (561–640 nm; for imaging mCherry, or Alexa-568-labeled microtubules) instead of far-red. Our three-color instrument is very similar except that the green, near-red, and far-red color ranges are projected onto three separate cameras. In either case, the different colors can be imaged simultaneously. Typically, we collect images at 10 frames/sec for ~200 sec. We have developed a series of semiautomated image analysis programs, written in LabView, to obtain the brightness, residence time, and mobility of individual particles bound to single microtubules. The basic analysis steps are straightforward and could also be implemented using ImageJ or Matlab. For convenience, this protocol describes the analysis of a single microtubule. Data from many microtubules across many experimental trials are needed to obtain robust conclusions that are independent of stochastic and trial-to-trial variability.