I am trying to independently analyze raw nanoparticle tracking analysis (NTA) data, but my diffusion coefficients calculated directly from x and y pixel values only moderately correlate with reported values and are consistently off by ~2x.
I attempted subtracting total video average x and y drift (i.e. pixels/frame) and per frame average x and y drift. I attempted fitting mean squared displacement vs time data by just averaging squared displacement, least squares regression with and without nonzero y-intercept, and least squares power law fit (even though anomalous diffusion should not be considered here) all with no better correlation. In fact, even for particle tracks of length 2 frames i.e. a single MSD and time interval, my values still only moderately correlate, depicted below. I have no idea what other variables could account for this variability other than particle intensity, but intensities are rarely used or reported and I have found no significant correlations.