Ultrahigh-resolution multicolor colocalization of single fluorescent probes

TLDR

The paper introduces an optical ruler that uses ultrahigh‑resolution colocalization of single fluorescent probes. The method employs two distinct fluorophore families—TransFluoSpheres and quantum dots—excited by a single laser, imaged with a nanometer‑step confocal microscope, and localized by fitting to the microscope’s point‑spread function. The technique achieves sub‑10‑nm distance accuracy in two‑dimensional colocalization of 40‑nm beads and 3–10‑nm quantum dots, bridging the scale gap between FRET and far‑field imaging from nanometers to micrometers.

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.