Ultrahigh-resolution multicolor colocalization of single fluorescent probes

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 energytransfer 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.A fter 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 organized structures, modular cellular machines that are self-assembled from a large number of interacting macromolecules and translocated from one cell compartment to another. To unravel the organization and dynamics of these molecular machines in the cell, a tool is needed that can provide dynamic, in vivo, three-dimensional (3D) microscopic pictures with nanometer resolution of individual molecules interacting with each other.Fluorescence microscopy can provide exquisite sensitivity down to the single molecule level for in vitro experiments (1-3). Moreover, it recently has been shown that single fluorophores can be detected in the membrane of living cells with good signal-to-noise ratio (S͞N) (4-6). What is not clear yet is whether single molecule fluorescence microscopy can provide the required spatial and temporal resolution. Technical challenges still to be met are (i) the synthesis of spectrally resolvable, bright, and stable fluorophores that can be coupled in vivo to macromolecules, (ii) the development of an easy-to-use and affordable instrument that permits high-resolution localization of individual point-like sources in 3D, and (iii) the ability to perform such measurements at a rate that is compatible with that of biological events.Recently, significant advances have been made in improving the spatial resolution of optical microscopy beyond the classical diffraction limit of light. These include (...

Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and Experiments

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Increasing the luminescence of lanthanide complexes

“…Intermittent dark states of individual nanocrystals can be a significant problem (36). According to Lacoste et al (36), ''They exhibit dark states that can span any duration from microseconds to seconds, sometimes accompanied by intermittent spectral jumps.…”

Quantum dots-based reverse phase protein microarray

“…The DNA-PKcs detection sensitivity limit achieved with the standard HRP-DAB assay is about 1 pM. The lysate has been further diluted in two-fold series down to 2 -11 dilution factor, effectively extending the dynamic range to be ~1000 (=2 10 ). The Qdot signals on the protein array have been detected as shown in Fig.…”

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