To track the movement of motor proteins with high precision in vitro, we use Fluorescence Imaging with One Nanometer Accuracy (FIONA). The image of a point-like fluorescent object is as wide as 250 nm in the visible region of the light because of the diffraction limit. The position of an object, however, can be localized very precisely by determining the center of its emission pattern. The precision depends on maximizing the photon detection per image and minimizing the noise factors. Yildiz et al. 2003 showed that millions of photons can be collected from a single molecule before it photobleaches. Organic dyes (Cy3, TMR, Cy5) were localized within ~100 msec, which enables to measure the step size of the motor proteins kinesin and cytoplasmic dynein. Using novel fluorescent probes, such as quantum dots, the time resolution of localization can be further improved to 10-20 msec.
Figure 1. FIONA. A. The Airy pattern of a diffraction-limited-spot in two dimensions. B. Fluorescence images of several single Cy3-DNA molecules immobilized on a glass surface. The data was taken with TIRF scope in 0.5 sec. C. Expanded view of one PSF with 2-D elliptical Gaussian curve fit (solid lines). The center of this PSF can be located to within 1.5 nm.
Figure 2. Kinesin stepping. Kinesin motors are labeled with a streptavidin coated quantum dot and their stepwise motion was tracked under TIRF. Above graph represents the stepping pattern of wild-type kinesin (WT) and engineered kinesin mutants with elongated neck-linker. As WT head takes regular 16 nm steps towards microtubule plus end, neck-mutants take more variable and bidirectional steps along.