committing

Author: AlexisStrain

CHEMOTAXIS/02_11_pf18.txt

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+### About: The code in the section is needed to find the Chemotaxis Indices (CI) of the Chemotaxis Lane Assay (CLA) Plate
+
+#### Protocol:
+DISCLAIMER: This is a shortened experimental protocol for quick reference. Please see publication for final methodology.
+1. 3D Print the CLA-plate in ABS.
+2. Set up the CLA-plate, so each lane has 1 ml of algae, at a 10^6 cell/ml, and the two reservoirs are filled with 1.5% solidified agar hydrogel. Suggested to do at least 3 replicates of each sample per experiment. 
+3. Image the plate every 5 minutes for 24 hours. Use a high-resolution camera with minimal "fish-eye" effect. 
+
+#### Data processing (imageJ):
+1. In ImageJ, open the images as a stack (file-> import -> image sequence).
+2. If the video moves you can align the stack to correct (plugin-> registration -> linear stack alignment with SIFT)
+3. Convert to 8-bit and invert.
+4. Rotate stack so that the lanes are exactly vertical to the screen y axis. Note which resevoir is at the top and bottom for next steps.
+5. Crop to the top and the bottom of the lanes. The resevoirs should be completely cropped out. 
+6. Open (file->open) the imageJ macro [y-axis_lane_profile](y-axis_lane_profile.ijm).
+7. Follow the macro prompts to get the intensity along the y-axis for each lane.
+8. The average of each lane will be saved as a "lane_number.txt" file in the same directory as the current stack, where lane 1 is the leftmost lane. 
+9. Rename each 'lane_number.txt' file as "DD_MM_sample_replicate number.txt" for each lane. 
+   
+#### Data processing (Jupyter Notebook):
+1. Move all of the "DD_MM_sample_replicate number(optional).txt" files from imageJ into the same directory as the Jupyter notebook [PUBLICATION_CHEMOTAXIS INDICES](https://github.com/AlaSummer/Chlamy-2024/blob/main/CHEMOTAXIS/PUBLICATION_CHEMOTAXIS%20INDICES.ipynb) for processed sample datasets.
+   - Note: sample data is provided 
+3. Follow the prompts in the notebook for further instructions to get the final indices.
+   
+

CHEMOTAXIS/02_12_CC-125.txt

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+macro "y-axis_lane_profile" {
+
+//called at end of macro by restoreSettings
+saveSettings();
+
+if (nSlices==1)
+   exit("Stack required");
+waitForUser("1. Change stack to 8-bit"+"\n"+"2.  Align to a horizontal and vertical axis manually."+"\n"+"3.Align stack if moves."+"\n"+"4.Crop to top of agars and sides of plate"+"\n"+"5.INVERT"+"\n"+"6.draw rectangle around ENTIRE channel");
+
+//save locations manually select output
+//Dir=getDir("Choose 2_LapseIt_output");
+
+
+//file save settings
+a=getDir("image");
+Dir=File.getParent(a);
+
+
+//gen settings:
+setBatchMode(false);
+Height1=getHeight();
+Width1=getWidth();
+Height2=(Height1/2);
+
+//finds pixel lenght of channel for square size
+ if (selectionType!=0)
+   exit("Rectangle selection required");
+Roi.getBounds(x1, y1, rectangleDimensionx, rectangleDimensiony);
+correctedDimensionx = rectangleDimensionx/2;
+
+
+//gets minLocs coordinates based on max intensity
+makeLine(0, Height2, Width1, Height2);
+run("Clear Results");
+profile = getProfile();
+minLocs= Array.findMinima(profile, 50);
+Array.sort(minLocs); 
+minLocsy=newArray(minLocs.length);
+minLocsy=Array.fill(minLocsy, Height2);
+makeSelection( "polyline", minLocs, minLocsy);
+lane=32;
+while (Roi.size !=lane) {
+   Dialog.createNonBlocking("Error");
+   Dialog.addMessage("Please select");
+Dialog.addNumber("Please Select lane number:", 32);   
+Dialog.addMessage("Current Number:"+Roi.size+"\n"+"To delete point, press alt and click");
+Dialog.show();
+lane=Dialog.getNumber();
+   }
+Roi.getCoordinates(minLocs, ypoints);
+
+//finding the average 1/3 position of every two maxima to find lane center more accurately
+arrayLength=(minLocs.length-1);
+finalPosition=newArray(arrayLength);
+for (dimension=0; dimension<(minLocs.length-1); dimension++) {
+   dimension2=dimension+1;
+   newPosition=((minLocs[dimension2]+2*minLocs[dimension]))/3;
+    finalPosition[dimension]=newPosition;
+   }
+ 
+
+   Dialog.createNonBlocking("Input Parameter");
+   Dialog.addMessage("Please input rate to sample frames in Stack.");
+   Dialog.addNumber("Sample every: ", 6);    
+   Dialog.show();
+   FrameSkipInterval =Dialog.getNumber();
+
+for (i=0; i<finalPosition.length; i++) {
+   x=finalPosition[i];
+   BB=i+1;
+   collectedValues="";
+   makeRectangle(x, y1, correctedDimensionx, rectangleDimensiony);
+   roiManager("Add");
+   N = nSlices;
+   for (k=1; k<=N; k+=FrameSkipInterval) {
+         run("Clear Results");
+         setSlice(k);
+         // to set the plot profile to run vertically, and take pixel average across x dimesion:
+         setKeyDown("alt");
+         profile = getProfile();
+         collectedValues=collectedValues+profile[1];
+      for (j=2; j<profile.length; j++) {
+         collectedValues=collectedValues+ "\t"+profile[j];
+         }
+      collectedValues=collectedValues+"\n"; 
+      }
+
+   //Save settings
+   file = File.open(Dir+"/lane "+BB+".txt");
+   print(file, collectedValues);
+   print("\\Clear");
+   File.close(file);
+   setSlice(1);
+   run("Clear Results");
+      }
+    }
+ }
+restoreSettings();
+roiManager("Show All with labels");
+}