Ultrahigh-resolution multicolor colocalization of single fluorescent probes

Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9461-6. doi: 10.1073/pnas.170286097.

Abstract

An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multicolor sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with 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. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near- and far-field imaging, spanning a range of a few nanometers to tens of micrometers.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Color
  • Energy Transfer
  • Fluorescence
  • Fluorescent Dyes / metabolism*
  • Microscopy, Confocal / instrumentation
  • Microscopy, Confocal / methods
  • Molecular Probe Techniques / instrumentation*
  • Sensitivity and Specificity

Substances

  • Fluorescent Dyes