Ultrahigh-resolution multicolor colocalization of single fluorescent nanocrystals.

A new method for <i>in vitro</i> and possibly <i>in vivo</i> ultrahigh-resolution colocalization and distance measurement between biomolecules is described, based on semiconductor nanocrystal probes. This ruler bridges the gap between FRET and far-field (or near-field scanning optical microscope) imaging and has a dynamic range from few nanometers to tens of micrometers. The ruler is based on a stage-scanning confocal microscope that allows the simultaneous excitation and localization of the excitation point-spread-function (PSF) of various colors nanocrystals while maintaining perfect registry between the channels. Fit of the observed diffraction and photophysics-limited images of the PSFs with a two-dimensional Gaussian allows one to determine their position with nanometer accuracy. This new high-resolution tool opens new windows in various molecular, cell biology and biotechnology applications.