Cell Motility Assay In This Assay, You Will Measure The Rate (in µm/sec

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Cell Motility Assay In this assay, you will measure the rate (in µm/sec) at which various Tetrahymena strains swim. In this assay you will collect 20 second videos of Tetrahymena swimming and use FIJI to measure its rate. An example of the type of video you will make can be found at (INSERT LINK). (This is a different protist captured on video at 10 frames/second). Applying your knowledge Shooting Tetrahymena videos Which objective will give you more data to work with? 1) Open Infinity Analyze on the laptop 2) Adjust the beam splitter on the microscope to send light Which condensor setting would make it easier to see Tetrahymena. Think about how to the camera. a condensor controls whether detailed 3) Expand the Camera Control Tab on the left. Adjust the features or large features of an image are able to be visualized. ? exposure time so that you can see Tetrahymena. Use the Area WB to set the background level. 4) Expand the Timelapse/Video tab on the left. To begin recording, select the the button to the right of the movie camera icon. After 20 seconds, select the recording button again to end the recording. 5) The video is automatically saved into your My Pictures directory as Image.Avi. Navigate to My Pictures and rename the Avi file. Otherwise the next recorded event will overwrite this video as another Image.Avi file. You should save your renamed Avi file onto your U drive. Measuring Tetrahymena swimming with FIJI With FIJI, the AVI video file is converted to a stack of images. You will make a Substack showing Tetrahymena swimming, and use Z Project to make tracks of the swimming. (The Z Project function looks at the pixel intensities at the same point in every image in a stack and makes a single image with either the minimum or maximum intensity values). Then you will make an Overlay of the track onto the substack. This will keep track of multiple Tetrahymena. 1) Open FIJI and import AVI file from My Pictures. File>Import> AVI 2) Use the slider bar at the bottom of the FIJI window t o move through the video. Determine which frames are of interest. 3) Make a substack. Image>Stacks>Tools>Substack. Save the substack on your U drive. 4) Make minimum intensity Z projection of the substack. Image>Stacks>Z Project. You should see the tracks of the Tetrahymena on the Z-Project. 5) Make an overlay of the Z-Project onto the substack. Select the substack, then: Image>Overlay>Add Image. Be sure that the image you add is the ZApplying Your Knowledge Project, set the opacity at 25%. When you play the How can you determine how many pixels are in a μm substack, you should Tetrahymena moving on their for the microscope with a particular objective? What stacks. can be used as a ruler in microscopy? 6) Use Set Measurements for measuring perimeters. Analyze>Set Measurements 7) Use Set Scale to set pixels/µm. Now you can use the Thinking Quantitatively How can you calculate the time between in frame in polygon tool to measure distances of the tracks with the substack. The standard video rate is 30 Analyze>Measure. frames/sec. Did the camera collect frames at this rate? 8) After measuring the lengths of the tracks in µms, calculate the rate of movement .