The standard diffraction limit of light is about 250 nm, meaning that you cannot “resolve” objects closer than this distance. Despite this, we have come up with a method to measure individual biomolecules with 1.5 nm spatial localization in x-y plane and 1-500 msec temporal resolution, using a technique we call Fluorescence Imaging with One Nanometer Accuracy (FIONA). We find that molecular motors, which are responsible for moving proteins around in the cytoplasm of the cell, walk—or even run—and in one case even seem to grow a long leg. Using a Hidden Markov Method (HMM), which is a technique able to extract signal from large background, we are also able to show that individual cargoes can be driven by more than one motor. In addition, we have recently extended the technique by using 2-photon (as opposed to 1-photon) excitation, with individual quantum dots. This has the advantage of seeing single molecules in 3-dimension, as opposed to the usual 2-dimension, and in being able to see in highly autofluorescent cells. Separately, we have managed to increase the resolution of 1-photon microscopy by at least 5-fold using a simple subtraction scheme.
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1000 MNTL, University of Illinois, Urbana-Champaign, IL