University:
Boston University
Course:
CAS CS 501 | Computer Science Practicum
Academic year:

2022
Views:

400

Pages:

9
Author:

numcore694sxk

Sample fluorescent image measurement  Open image TfnR-A555-Rab11-A647-34101.tif  The red is transferrin receptor, a carrier protein  The green is Rab11, an enzyme  Imaged to investigate when these molecules are colocalized.  Adjust the B&C as you like (don’t press Apply)  We will measure the intensity, location, and size of the red fluorescent transferrin receptors. Red Channel  Strategy:  Remove the background (out of plane light)  Segment the red channel image into separate blobs for measurements.  Make the measurements on the original red channel image.  Save the measurements to a file.  Split the RGB image into separate channels: ImageColorSplit Channels  Close the green & blue channels  Click ImageDuplicate to make a copy in a new window. Background  Adjust B&C again.  Click ProcessSubtract Background  Apply a 12 pixel radius rolling ball subtraction  Note that there are many other ways to do a background subtraction… Thresholding  Now apply a threshold to select brighter regions  Click ImageAdjustThreshold  Which threshold to use?  Try a few!  Choose the Otsu threshold, click Apply. Create ROIs  ROI: “Region of Interest”  ImageJ can handle many ROIs at once using the ROI Manager tool.  Click AnalyzeAnalyze Particles.  Set the size to 4-2500  Set Show to None  Uncheck everything but Add to Manager  Click the OK button.  The ROI manager will open. Measurements  Click the original red channel image.  In the ROI manager, click in the list of ROIs and press Ctrl-A to select them all.  Click the Add button.  Uncheck the Labels option.  The ROIs will be highlighted in the original image.  Click AnalyzeSet Measurements, choose some measurements to make. Measurements cont’d  In the ROI manager, click the Measure button.  This will measure all ROIs separately.  Click AnalyzeMeasure to measure all the ROIs as a single area  Measurements will be displayed in a new window.  The measurements can be saved or copied using the File and Edit menus. Adding Plugins     Click HelpUpdates Click the Manage Update Sites button Click a site or too, then click the Close button. New plugins can now be installed. Where to go from here…  ImageJ documentation is online.  Web searches will turn up many years worth of online discussions on ImageJ features.  A vast array of plugins and macros are available.  And you can write your own!’  For processing large batches of images version 1.51g of ImageJ is available on the SCC using the fiji/2015.12.22 module.