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Post-Hoc Vibration Mitigation for Single-Molecule Tracking and Diffusion Measurements in Lipid Membranes

ABSTRACT Single-molecule tracking algorithms and analyses that use particle trajectory information rely on the accurate determination of particle positions to produce a meaningful interpretation of molecular dynamics. However, artifacts arising from vibration can sometimes distort or entirely mask the processes being studied. Here we describe a straightforward implementation of the Lucas-Kanade stabilization algorithm that can remove the impact of instrument-related vibrations on molecular diffusion measurements after a data set has been collected. Using fluorescently derivatized αHL on supported lipid bilayers in conjunction with computer simulations of 2D diffusion, we report that post-hoc stabilization can be effective in removing both simple and complex noise sources. The effectiveness of vibration mitigation is a function of the relationship between the vibration amplitude and the magnitude of the diffusion constant. For diffusion constants in the range of typical membrane proteins (e.g., 10−9 to 10−7 cm2s−1), vibrations that produce errors in excess of 100% can be reduced to producing errors less than 5%. The algorithm is effective even when the relative magnitude of the vibration is high and can be especially useful in experiments where vibrations cannot be entirely removed by experimental means alone. Supplemental materials are available for this article. Go to the publisher's online edition of Spectroscopy Letters for the following free supplemental resources: Video content illustrating single–molecule tracking capability, output from the single–molecule simulator, and the impact of post-hoc vibration stabilization.