Running ImageJ particle trackerA. F. Emery, 5/21/2015

Analyzing the Bubble movies involves 2 stepsA) Making the ‘stack.tif’ file. This contains all of the information in the movie frames.B) Analyzing the information in the ‘stack.tif’ file

Making the ’stack.tif’ file1) Make sure that you know where your .bmp files are located2) Open ImageJ. The following window will appear. A progress bar also appears at the

bottom—in this example it says ‘Detecting Particles in Frame 15144’

3) Click on Plugins->macros->install go to the ImageJ folder. The ‘macros’ folder will open automatically. Scroll down the list, click on ‘tiff_to_stack.txt’ and then Open. The progress bar will show ‘1 macro installed.’ THIS STEP MUST BE REPEATED EVERY TIME THAT YOU WANT TO RUN make a ‘stack.tif’ file.

4) Click on Plugins->macros->batch convert. A window will open requesting the location of the .bmp files. Click on the folder containing the bmp files, (the window will not display any of the .bmp files so don’t be confused). Select the folder. You will then be asked where the ‘stack.tif’ file should be located. I usually just leave it in the same folder as the .bmp files. Click ‘select’. ImageJ will immediately start creating the ‘stack.tif’ file and the converter will then run as shown by the moving bar on the ImageJ tool bar. After it finishes writing (it will stop but not tell you that it has finished)

5) Click on ‘File’, open the folder containing the ‘stack.tif’ file and select the file. The file thumbnail will show the first frame of the movie. The program will then process the file, telling you how many records it has read.

6) A screen shot will then come up showing the first frame. You can move the slider on the bottom of the screen shot to see all of the frames continuously.

7) Now click on Plugins->Particle Detector & Tracker -> Particle Tracker. The progress

bar will say that it is calculating a histogram and the above dialog will appear. Enter the radius of the bubbles that you wish to track and the link range. The link range specifies how many consecutive frames that a particle must be in for a trajectory to be created. Click ‘OK’ and the progress bar will show what frames are being analyzed. This step can take a long time.

8) I GENERALLY ANALYZE NO MORE THAN 1000 FRAMES AT A TIME., The program will analyze every .bmp file in the folder so you may want to set up several folders in order to analyze specific time intervals in the movie.

9) After all the .bmp files have been analyzed, the following window will appear.

telling how many trajectories were found..

10) Click on ‘Visualize All Trajectories’.

The trajectories in the 1st frame will be shown (note that in this screen shot we see frame 1 of 292).

11) You can move the slider on the tool bar and watch the trajectories as you go through all of the frames.

12) If you click on ‘Filter Options’ you can change the link value. A new result page will come up and a new trajectory picture. Clicking on the ‘Results’ page will show the new number of trajectories. It is a good idea to try different link values to get an idea of how long trajectories are so that you can use this when running the Matlab program “Trajectories’. Here the 1674 trajectories with a link range of 2 was reduced to 187 for a range of 20.

13) Once you have created a ‘stack.tif’ file, you can always restart ImageJ and begin at step 5 14) If you choose too many bmp files to analyze, you will probably run out of memory. In

this case you will need to select a smaller number when making the ‘stack.tif file’.15) Finally you need to save the ‘Full Report’. This will list every trajectory found based

upon the radius and link range specified initially in step 7. It is not affected by any filter options.

16) Now run the Matlab program ‘Trajectories’ to see the final results. The inputs to the program are the link range and trajectory length desired. Type help Trajectory to read

about them.

17) Trajectory gives several plots. Here is a plot of trajectories with each trajectory labelled with its number placed at the beginning of the trajectory.

Experiment with different link ranges and trajectory lengths to get a good idea of where the bubbles were going. In this movie, most were going out of the room by flowing under the door threshold.

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Trajectory