3D particle tracking using IDL -- John C. Crocker and Eric R. Weeks
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If you want better discrimination in Z, there are a few options. First, you can take your image slices in Z closer together. Typically we do 0.2 micron spacing in Z and thus we're stuck with an error that's not much better than 0.2 microns. Second, if you have a dilute system of particles, then you can increase your diameter in Z and get better subpixel resolution. (This works in X and Y as well). However, this only works if you have particles spaced far apart, otherwise you'll grab information from other particles. This will result in less apparent pixel-biasing, but it's misleading, and won't help your particle location.

I hope to write more about this later. A couple quick comments, though. First, the better your resolution, the sharper your pair correlation function will look. In general, if you do something to poorly pretrack your data, it will only make the pair correlation function look worse, in the sense that the height of the first peak will be diminished. Second, if you track your data, you can calculate the mean square displacement (MSD). The better your resolution, the lower the MSD will be. Again, hard to do a bad job pretracking without having it increase the MSD. Be sure to check all three directions , , .

One last comment: sometimes you can do extremely well at pre-tracking, as defined by these measures, but only by throwing out any and all even slightly ambigious particles. This is up to you. If you want a nice looking mean square displacement curve, maybe this is OK; but for the pair correlation function, it seems like one ought to consider as many particles as you think are real, even if their positions aren't terribly accurate. As always, tracking is a bit of an art and you can make a judgement call on the tradeoff: track every particle, or fewer particles better. I usually prefer the former.

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