User:Bohao Liu/Notebook/Research/2014/04/30

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HOG Experiments : wild - type controls and Image Analysis Parameters

Objective

  • Make sure I can do HOG experiments again
  • Confirm Wild-type values and also expand the frequencies tested
  • Confirm the protocol: specifically that waiting 30 minutes between each set of oscillations is enough to ensure that the cells return to normal
  • See if the cells are growing

Experiment

  • Cells OD @ 10 = .453
  • Started flow at 10:45 am
  • Started experiment at 11:15 am

Settings

  • 30 second half-period
  • 5 second imaging = 6 images per cycle
  • 5 minute total time = 5 complete periods
  • ND=2
  • mCherry = 200 mS exposure
  • GFP = 200 mS exposure
  • DIC

Image Analysisp

  • for windows sorting :
    • for /l %x in (1,1,5) do MD well%x
    • for /l %x in (1,1,5) do MOVE col*t*xy%xc*.tif well%x


//Sets Measurements run("Set Measurements...", "area mean centroid redirect=None decimal=3");

//Asks for directory dir = getDirectory("Choose a Directory"); folderList = getFileList(dir);

for (i = 0; i < folderList.length; i++) { if (endsWith(folderList[i], "/")) { wellList = getFileList(dir+folderList[i]); for (j = 0; j < wellList.length; j++) { fileList = getFileList(dir+folderList[i]+wellList[j]);

//Now starts actual analysis filename = dir+folderList[i]+wellList[j]+fileList[0];

//Opens image sequence, c1 is mCherry run("Image Sequence...", "open=filename starting=1 increment=1 scale =100 file=[] or=c1. sort"); run("Subtract Background...", "rolling=50 stack"); setAutoThreshold("Otsu dark"); run("Threshold..."); run("Convert to Mask", " black"); run("Erode","stack"); run("Despeckle","stack"); run("Analyze Particles...", "size=25-100 circularity=0.50-1.00 show=Nothing add stack"); close(); run("Image Sequence...", "open=filename starting=1 increment=1 scale =100 file=[] or=c2. sort"); run("Subtract Background...", "rolling=50 stack"); roiManager("Measure");

//CHANGE ME****************** saveName = "C:\Users\NIKON\Documents\Bohao\HOG\Results"+substring(folderList[i],0,lengthOf(folderList[i])-1)+substring(wellList[j],0,lengthOf(wellList[j])-1)+"nuc.txt"; saveAs("Results", saveName); roiManager("Reset"); run("Clear Results"); close(); }

} }





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