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.

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 (sm) (figure from Yildiz et al. Science 2003).

|