User Manual 1
Tracking Neutrophils with PhagoSight 1
To track neutrophils, Matlab requires a series of functions and routines that in some cases call other functions or subfunctions. The data input should be a directory where you should have a series of directories (one for each time frame) with tiff files, one for each slice of the data. This is format how volocity exports the data. So before using Matlab you need to export the data from volocity.
It is important to keep names simple, as the tracking will create several folders related to the original folder, try to avoid spaces and special characters such as &, ^, %, &, +, etc. It is better use only a to z, A to Z and numbers. So, if you have a folder with the name "images", the following folders will be created:
- % example of the output directories :images_mat_Or <- original data in matlab format
- % example of the output directories :images_mat_Re <- data reduced in size
- % example of the output directories :images_mat_La <- labelled data
- % example of the output directories :images_mat_Ha <- Handles, this folder holds the results
The first step will read the data from the TIFF files and transform it to Matlab, the function "readNeutrophils" will then in turn write on a new root folder a series of directories with the data in matlab format.
Then, to reduce computational complexity by averaging neighbouring pixels in a uniform pyramid, 2 levels would reduce from 1000x1000 to 250x250 reduceNeutrophils will read the folder and then iteratively reduce size in each frame and save setNeutrophilHandles determines the dimensions of the data set and the *thresholds* used to segment "reduceNeutrophil" will threshold and label the neutrophils. To increase the accuracy for the segmentation a double thresholding is performed, small blobs are removed as noise.
All this pre-processing is required to perform the tracking with the function "trackNeutrophil" The final data is stored inside handles, handles.finalNetwork and handles.nodeNetwork have all the data, handles.nodeNetwork has the following structure:%[ X Y Z distancetoNext timeFrame ID Parent Child - Volume Label KeyholeRegion Track FinalLabel BoundingBox(X init Y init Z init X Y Z) ]
The syntax of the matlab command for the tracking is:[handles]= preProcessingNeutrophils(dataInName,numLevelsToReduce);
dataInName : is the name of the folder where the subfolders where the tiff files are located, you can either pass this name or select it later. numLevelsToReduce : this controls the reduction of the data, the default value is 2 that will reduce the images from 1000 x 1000 to 250 x 250. handles keeps the results.
So, to process a folder called "NeutropTest" (which contains 9 folders with tiff files) the easiest way to analyse it is to call the function with no parameters:
This will open a window from which a folder can be selected:
So, for example, to open the folder "NeutropTest" you should navigate to the corresponding folder:
Once you select the folder, the processing will start and as the data is processed this will appear in the command window:
Read tiff images from folders and save as matlab data Read matlab files from folder, reduce and save in a new folder *_mat_Re
The data will now be saved in one file (T00001.mat, T00002.mat, T00003.mat, ... inside the _mat_Re folder) for each time frame. The file contains the 3D structure corresponding to all the images of the frame. These slices can be of fluorescent or DIC channels, so the algorithm will now open a new window to determine the structure. The algorithm will count the number of images (or slices or levels) and then try to distinguish between DIC and fluorescent. DIC will spread the intensity in the middle of the range, whilst fluorescent will concentrate in the low levels (to the left). For instance, a case of 6 levels, 3 green fluorescent and 3 DIC will look like this:
While a case of 40 Green and Red channels would look like this:
The algorithm cannot distinguish between Green and Red Channels, so it will assume that all fluorescence is green,
so you need to correct this manually in the appropriate window.
Once you have selected the correct distribution of the levels, the algorithm will continue:
Immediately after selecting the appropriate levels for the fluorescence and DIC, the threshold levels are automatically selected. In some cases, these levels need to be manually verified. This is done with the first time point of the data.
The algorithms will select the low and high thresholds and a group of windows will be opened to display:
(1) The maximum intensity projection (the highest value each pixel(row,column) can have for each slice down the Z-stack) and the pixels of this projection that are above the two thresholds. The region of highest intensity (strongest fluorescence) will be selected to zoom in.
(2) A detailed region where the highest intensity is located, and
(3) A 3D plot of the neutrophils in that region. A red surface will indicate the low threshold and the blue surface will indicate the high threshold. This plot is three-dimensional and you can rotate it to find the best view for the neutrophils in question.
If you want to change the region to plot (and thus analyse some interesting neutrophils, two that are very close to each other for instance), or you want to modify the thresholds, you can do that at this moment through the command line, where you will be asked if you want to change something. If you do not want any changes, just press enter. Otherwise, type 'y' and enter. Then you will be prompted for the region to plot (initial Row, final Row, initial Column, final Column) and the thresholds. If you do not want to change any of them, press enter, otherwise, type the new value and press enter.
Do you want modify plot or threshold levels? Y/N [n]: y
After typing 'y' and return you are prompted to change parameters. It looks like there are two neutrophils very close to each other around the region of [210-260] in the rows and [510-570] in the columns (that is to the right of the zoom region). Thus those parameters are changed:
Type the new values for the following parameter or press RETURN to leave unchanged Low threshold =512.6471;800 High threshold =756.2059;1200 Initial row to plot = ; Final row to plot = ; Initial column to plot = ; Final column to plot = ;
Now it is time to modify the thresholds. It looks like the neutrophils are merged into one single cell at both thresholds, therefore, let's try with higher levels, say 880 and 1100 (you may need to go through several iterations to find the optimal thresholds):
Type the new values for the following parameter or press RETURN to leave unchanged Low threshold =512.6471;880 High threshold =756.2059;1100 Initial row to plot = ; Final row to plot = ; Initial column to plot = ; Final column to plot = ;
Notice that all other parameters remain the same.
Now that the neutrophils appear separate from each other, the process can continue. Just press enter at the prompt to keep the same values and continue with the process:
Read reduced matlab data, Threshold, Label and save in a new folder *_mat_La Split Large Neutrophils and re-save in same folder *_mat_La Initial Tracking process Keep the disappearing cells and re-save in the same folder *_mat_La Second Tracking process Split merged cells and re-save in a the same folder _mat_La Final Tracking process