We review recent advances in the development of colloidal fluorescent semiconductor nanocrystals (a class of quantum dots) for biological labeling. Although some of the photophysical properties of nanocrystals are not fully understood and are still actively investigated, researchers have begun developing bioconjugation schemes and applying such probes to biological assays. Nanocrystals possess several qualities that make them very attractive for fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer fluorescence lifetime than organic fluorophores and negligible photobleaching. On the down side, their emission is strongly intermittent ("blinking") and their size is relatively large for many biological uses. We describe how to take advantage of nanocrystals' spectral properties to increase the resolution of fluorescence microscopy measurements down to the nanometer level. We also show how their long fluorescence lifetime can be used to observe molecules and organelles in living cells without interference from background autofluorescence, a pre-requisite for single molecule detectability. Finally, their availability in multicolor species and their single molecule sensitivity open up interesting possibilities for genomics applications.
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 (...
We present single-pair fluorescence resonance energy transfer (spFRET) observations of individual opening and closing events of surface-immobilized DNA hairpins. Two glass-surface immobilization strategies employing the biotin-streptavidin interaction and a third covalent immobilization strategy involving formation of a disulfide bond to a thiol-derivatized glass surface are described and evaluated. Results from image and time-trace data from surface-immobilized molecules are compared with those from freely diffusing molecules, which are unperturbed by surface interactions. Using a simple two-state model to analyze the open and closed time distributions for immobilized hairpins, we calculate the lifetimes of the two states. For hairpins with a loop size of 40 adenosines and a stem size of either seven or nine bases, the respective closed-state lifetimes are 45 +/- 2.4 and 103 +/- 6.0 ms, while the respective open-state lifetimes are 133 +/- 5.5 and 142 +/- 22 ms. These results show that the open state of the hairpin is favored over the closed state of the hairpin under these conditions, consistent with previous diffusion fluorescence correlation spectroscopy (FCS) experiments on poly(A)-loop hairpins. The measured open-state lifetime is about 30 times longer than the calculated 3 ms open-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS study for hairpins in diffusion with 12-30 thymidines in their loops. As predicted, the closed-state lifetime is dependent on the stem length and is independent of the loop characteristics. Our findings indicate that current models should consider sequence dependence in calculating ssDNA thermostability. The surface immobilization chemistries and other experimental techniques described here should prove useful for studies of single-molecule populations and dynamics.
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