datafiles_key.txt

This document describes the procedure and parameters for processing the raw 
microscope data in ImageJ using the Mosaic plugin. 

1) The raw .tiff image stack is imported into ImageJ
    File > Open > {select the datafile}

2) Adjust the grayscale threshhold. Since the images are 8-bit grayscale, we 
can set a grayscale value to make all lower values go to minimum and all 
higher values go to maximum. This step effectively subtracts the background 
of from the spheres we want to track. 
    Image > Adjust > Threshold
A window appears with controls to manipulate the threshold values. The 
threshold can be manually adjusted, but we chose to use one of the "smart" 
settings. This setting picks a threshold value that maximizes one of the 
several possible performance metrics. Among those listed were: "Mean," 
"MaxEntropy," "MinError," "Minimum," "Intermodes," and many others. Using 
of the preset threshold metrics also prevented unintential biased sampling 
from affecting the background selection if we chose the value ourselves. We 
chose the "MaxEntropy" setting after examining the background subtraction 
performance of each setting. MaxEntropy appeared to chose the threshold value 
value that best included the actual plastic spheres while introducing minimal
unwanted noise. This selection was made, and with all three of the boxes 
unselected, the "Apply" button is pressed. 

3) Select the Mosaic plugin
    Plugins > Mosaic > Particle Tracker 2D/3D

    Use the parameters:
    radius = 10 (pixels, for 2 micron spheres), 6 (for 1 micron spheres)
    cutoff = 3.0
    per/abs = 0.5 (for 2 micron spheres), 0.1 (for 1 micron spheres)
    link = 5
    displacement = 10

    Press "Ok" to start the analysis. This will take some time.