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Unformatted text preview: Single molecule restriction digest Tracking Tethers Hernan Garcia April 27, 2009 1 Assignment Follow the tutorial described in the remainder of this write up for one frame of your choice taken during the single-molecule session. Create a gallery of some of the images generated in intermediate steps of the analysis: Im, ImThresh, ImMask, and ImOverlay . Also, make sure to show the before and after pictures if you’re cropping the images. Once you’ve worked out a strategy to find the beads, analyze your whole movie (or skip every two or three frames if your movie is too long) and generate a plot of the number of beads found as a function of time. You might want to show some frames of the movie corresponding, for example, to the first, last and some middle frame. 2 A little tutorial Our objective is to find the number of tethered beads remaining as a function of time after flowing in restriction enzyme. Even though we don’t expect each group to have comparable results in terms of the time scales obtained, we think of this exercise as a good way to deepen your knowledge of image analysis using Matlab. First, we need to decide on a strategy to work with the images. You should move all the TIF files acquired with Micro-Manager to a folder, where we’ll be working from. Before writing the part of the code that deals with loading and analyzing multiple images, it is always a good idea to load only one in order to optimize your choice of parameters. We then start by loading one of the images. In the case of the little movie I took, the image I choose to optimize my analysis with is called img 000000000 Snap 000.tif . Let’s load it and display it as follows 1 Im=imread(’img_000000000_Snap_000.tif’); imshow(Im,) In some cases you might decide that you have too many beads or you might realize that some part of your field of view is out of focus. If you want to keep just one section of your image this can be easily done remembering that an image in Matlab is just a matrix. If you want to find out the size of this matrix you just have to do size(Im) In my case I got two numbers:...
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- Fall '09
- RNA, Want, ImLabel