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Data Analysis for Total Internal Reflection Fluorescence Microscopy.

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.