Ultrahigh resolution multi-color colocalization of single fluorescent probes

Abstract

A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Ser. No. 60 / 228,107, filed Aug. 24, 2000, the contents of which are incorporated herein in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under Grant (Contract) No. DE-AC03-76F00098 awarded by The United States Department of Energy. The government has certain rights to this invention.BACKGROUND OF THE INVENTION [0003] Following the completion of the human genome project, the cataloging of all gene sequences and the acquisition of high-resolution structures of proteins and RNAs, future biological investigations will focus on how the fundamental cellular building blocks interact with each other. Another important issue will be to determine their precise locations in space and time in an attempt to decode and lay out the cell machinery and circuitry. Indeed, many vital functions of the cell are performed by highly...

AI Technical Summary

Benefits of technology

The patent text discusses the limitations of various methods for improving the resolution of optical microscopy. These methods have limitations such as requiring subsequent signal processing, difficulty in implementing for hydrated samples, and constraints on sample size. The text also mentions attempts to correct or overcome these limitations, but acknowledges that none of these methods perfectly corrects the imperfections of far-field optics. The technical effect of the patent text is to provide a solution that overcomes the limitations of existing methods and improves the resolution of optical microscopy.

Problems solved by technology

While opening new frontiers in microscopy, each of these methods has particular limitations.

For example, deconvolution algorithms do not have unique solutions, require subsequent signal processing, and have limited ability to compensate for aberrations.

Moreover, it is very demanding to operate and difficult to implement for hydrated samples.

PSF engineering by STED requires an ultra-fast oscillator and amplifier system, and is not easily expandable to multicolor probes.

More generally, aside from NSOM, these super-resolution approaches still suffer from basic limitations of far-field optics, i.e. spherical and chromatic aberrations that can never be fully corrected for all wavelengths, and all have to tackle with the registration of separately acquired color planes.

However, none of these approaches perfectly corrects these imperfections.

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