<title>Ultrahigh-resolution multicolor colocalization of single fluorescent nanocrystals</title>

Abstract

A new method for in vitro and possibly in vivo 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-spreadfunction (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.

Properties of Fluorescent Semiconductor Nanocrystals and their Application to Biological Labeling

“…A detailed description of the custom-made microscopes used in these studies is given elsewhere [42,78,79].…”

“…The uncertainty in this distance measurement was calculated using the bootstrap method [84]. It yielded a final distance of 25 nm and a "2σ" error bar of 13 nm, comparable to the error bar obtained for non-blinking beads [42,79]. Strong blinking significantly affects the reliability of the fit, and a careful analysis of the validity of the bootstrap estimation has in principle to be performed for each blinking statistics.…”

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“…Quantum dots are nanometer-sized semiconductor crystals with special optical qualities, although at present the 'blinking' of quantum dots renders them unsuitable for some single-molecule experiments [34]. Whether blinking can be eliminated remains an open question.…”