Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch

Abstract

We demonstrate a novel all-optical switch consisting of two molecules: a primary fluorophore that can be switched between its fluorescent and dark states by light of different wavelengths, and a secondary chromophore that facilitates switching. The interaction between the two molecules exhibits a distance dependence much steeper than that of Förster resonance energy transfer. This enables the switch to act as a ruler with the capability to probe distances difficult to access by other spectroscopic methods, thus presenting a new tool for the study of biomolecules at the singlemolecule level.Single-molecule technologies have made a significant impact in many areas of science and engineering, ranging from electronics and photonics to molecular and cell biology. The detection of single biomolecules has transformed the way we study biological systems [1][2][3][4][5][6][7]. It is now evident that proteins, RNA and even DNA molecules are much more dynamic than previously thought, exhibiting structural dynamics essential for their function. Experiments that probe the behaviour of individual molecules in real time have proven to be ideal for characterizing these structural dynamics and their functional implications. Förster resonance energy transfer (FRET) is the most widely adopted technique for studying the conformational dynamics of individual biomolecules [6][7][8][9]. The FRET efficiency ε between a donor and an acceptor fluorophore depends on the intermolecular distance R, with ε = 1 / (1 + (R / R 0 ) 6 ). The Förster radius R 0 is typically 4 -6 nm for donor-acceptor pairs that are sufficiently bright to be detected at the single-molecule level. Therefore, single-molecule FRET is a useful probe of conformational changes on the order of several nanometers [6][7][8][9][10][11][12]. Few methods exist for the study of structural dynamics at shorter length scales. A promising technique for monitoring angstrom-scale distances, based on electron transfer, has recently been demonstrated using a flavin enzyme [13]. However, the length scale of 1 -3 nm remains difficult to access by non-invasive single-molecule techniques.In this work, we demonstrate a single-molecule optical switch which can function as a shortrange spectroscopic ruler to probe distances down to 1 nm. The molecular switch is based on a cyanine dye, Cy5, which we designate as the primary switch molecule, and a secondary chromophore, Cy3, which facilitates switching of the Cy5. For the purpose of single-molecule †To whom correspondence should be addressed. E-mail: zhuang@chemistry.harvard.edu. * These authors contributed equally to this work detection, the Cy5 and Cy3 were attached to opposite strands of a double-stranded DNA molecule and immobilized on a fused quartz surface through a streptavidin-biotin linkage (Fig. 1A). Molecules immobilized on a polyethylene glycol (PEG)-coated surface yielded similar results. Individual Cy5/DNA/Cy3 constructs were imaged in Tris buffer (pH 7.5) with an oxygen scavenging system to reduce the photobleaching rate...

Electromagnetically induced transparency with superradiant and subradiant states

“…EIT involves at least three levels and naturally three-level atoms are used in most cases. However, proper three-level structures are not available in some optical systems, such as in atomic nuclei [11][12][13] and biological fluorescent molecules [14], in which EIT can have important applications once realized. Interestingly, it has been shown that even with only two-level systems, EIT-like spectra can be achieved by locally addressing the atomic ensembles [15][16][17].…”

Optical control of resonance energy transfer in quantum dot systems

“…2 (c)). In this case, optical switching can be produced by the throughput of a single off-resonant beam [6], [7]. d) Distance dependence: Owing to the (R) −6 distance dependence on the total transfer rate [3], lower relative Laser intensity (Wm -2 )…”

Optical imaging of nanoscale cellular structures

“…However, that number still suffices for a ten-fold resolution enhancement, and FPs are easier to use and ideally suited for in vivo experiments because they are expressed by the cell itself (Hess et al 2007;Shroff et al 2008). Synthetic dyes, by contrast, require specific buffer conditions involving high concentrations of reductants for photoswitching, and additional labeling procedures that limit their application to fixed specimens (Bates et al 2005;Heilemann et al 2005). PALM with independently running acquisition (PALMIRA) ) is a simplified version of PALM that uses only a single laser line, and direct STORM (dSTORM) is a simplified version of STORM that avoids the second dye molecule for activation (Heilemann et al 2008).…”

“…In general, synthetic dyes are more photostable than fluorescent proteins and, therefore, provide a higher localization precision. For example, ∼6,000 photons per molecule were reported per switching cycle using the photoswitchable fluorophore pair Cy3-Cy5 (Bates et al 2005(Bates et al , 2007, whereas tdEosFP, one of the brightest PAFPs for PALM imaging, yields only ∼2,600 photons per molecule (Shroff et al 2007), and monomeric FPs often only a few hundred photons (Fuchs et al 2010). However, that number still suffices for a ten-fold resolution enhancement, and FPs are easier to use and ideally suited for in vivo experiments because they are expressed by the cell itself (Hess et al 2007;Shroff et al 2008).…”