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%xfor /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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Project name
