Single-molecule high-resolution imaging with photobleaching

Abstract

Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a ''gap'' in the ability of optical techniques to measure distances on the 10-to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10 -20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.T he advent of single-molecule imaging has enabled a revolution in the measurement of the physical parameters underlying biological processes (1). However, there are limitations on the length scales over which single-molecule imaging can be used to measure distances. Conventional far-field microscopy techniques are limited by the Rayleigh criterion (2) to resolving distances greater than ϳ200 nm. Some recent techniques have been developed to circumvent this limitation (3, 4), but they are technically demanding and at this point of limited applicability to biological systems. Fluorescence resonance energy transfer (FRET) can be used to measure distances much smaller, on the order of a few nanometers. However, because of the strong distance dependence of energy transfer, FRET is limited to measuring distances less than ϳ10 nm (5). As a result, there is a ''gap'' in the resolution attainable by single-color optical spectroscopy, making it difficult to measure separations between 10 and 200 nm. Many biological objects of interest are on this scale, including DNA structures, macromolecular complexes, and motor proteins. Although it is possible to measure distances on these scales by using two or more dyes of different colors (6, 7), this technique presents its own problems, such as how to achieve heterogeneous labeling, and calibration of the distance registration between different color wide-field images.Recently, with the introduction of low-noise high-quantumyield charge-coupled device (CCD) cameras, it has become practical to localize individual fluorescent dyes at subwavelength scales (8-10). We recently demonstrated the ability to determine the positions of single molecules at room temperature with a precision of 1...

Conformation and physics of polymer chains: a single-molecule perspective

“…For other applications, the chain can be labeled with multiple dyes, such as by capping both ends [70,71] or labeling with three dyes along the chain [17]. Nanometer-scale localization of the dyes at both ends by super-resolution microscopy then gives the end-to-end distance directly for a single polymer chain [70], information that is unavailable by any other non-invasive analytical method. If the two dyes are spectrally distinct, the end-to-end distance of the chain can be determined by the fl uorescence resonance energy transfer between them [72].…”

“…In solutions or melts, the diff usion of labeled chains can be followed by direct fl uorescence imaging in the case of slow diff usion or by single-molecule fl uorescence correlation spectroscopy in the case of fast diff usion [69]. For other applications, the chain can be labeled with multiple dyes, such as by capping both ends [70,71] or labeling with three dyes along the chain [17]. Nanometer-scale localization of the dyes at both ends by super-resolution microscopy then gives the end-to-end distance directly for a single polymer chain [70], information that is unavailable by any other non-invasive analytical method.…”

Plasmonic Control of Radiative Properties of Semiconductor Quantum Dots Coupled to Plasmonic Ring Cavities

“…their photostability). In particular, at near single-emitter level, organic dyes photobleach quickly [22][23][24][25] and quantum dots (QDs) tend to blue-shift and also change their switching and radiative decay behavior after prolonged exposures to high intensities. [26][27][28] The small sizes of the hotspots of dimer nanoantennas limit the interaction to just a few emitters, effectively forcing the system towards such a single-emitter level.…”

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

“…7). In practice, this can be achieved more readily by comparing the images before and after the photobleaching of one of the molecules [22,23]. In the second case, very high positional accuracies have been reported (down to 1 nm) [24,25].…”

“…Distances between molecules separated by sub-resolution spacings can be measured by comparing the diffraction patterns as the molecules photobleach. For example, the distances between fluorophores bound to DNA and separated by 10 nm have been measured [22]. Photobleaching (and subsequent recovery) can also be used to investigate the stoichiometry of multi-protein complexes and the turnover of subunits, as was recently done for the bacterial flagellar motor [32].…”