Michel Orrit

Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod

Existing methods for the optical detection of single molecules require the molecules to absorb light to produce fluorescence or direct absorption signals. This limits the range of species that can be detected, because most molecules are purely refractive. Metal nanoparticles or dielectric resonators can be used to detect non-absorbing molecules because local changes in the refractive index produce a resonance shift. However, current approaches only detect single molecules when the resonance shift is amplified by a highly polarizable label or by a localized precipitation reaction on the surface of a nanoparticle. Without such amplification, single-molecule events can only be identified in a statistical way. Here, we report the plasmonic detection of single molecules in real time without the need for labelling or amplification. Our sensor consists of a single gold nanorod coated with biotin receptors, and the binding of single proteins is detected by monitoring the plasmon resonance of the nanorod with a sensitive photothermal assay. The sensitivity of our device is ∼700 times higher than state-of-the-art plasmon sensors and is intrinsically limited by spectral diffusion of the surface plasmon resonance.

Room-Temperature Detection of a Single Molecule’s Absorption by Photothermal Contrast

Hot Enough to See Over the last decade, detection of fluorescence from individual molecules has allowed for increasingly detailed probing of biochemical reaction mechanisms. The key advantage of fluorescence detection is the absence of background; the signal appears as a glowing point in a void. However, not all molecules fluoresce, and so alternative detection methods are needed. Gaiduk et al. (p. 353) now show that a photothermal detection scheme can resolve absorption events by individual molecular dyes that exhibit poor fluorescence efficiency. The technique relies on each molecules release of heat to the surrounding solvent after light absorption, an energy dissipation mechanism that is enhanced as fluorescence efficiency declines. The solvent heating alters the local refractive index just enough to scatter a portion of a probe beam backwards, revealing the absorption. Single molecules have been detected through the heat they release after absorbing light. So far, single-molecule imaging has predominantly relied on fluorescence detection. We imaged single nonfluorescent azo dye molecules in room-temperature glycerol by the refractive effect of the heat that they release in their environment upon intense illumination. This photothermal technique provides contrast for the absorbing objects only, irrespective of scattering by defects or roughness, with a signal-to-noise ratio of ~10 for a single molecule in an integration time of 300 milliseconds. In the absence of oxygen, virtually no bleaching event was observed, even after more than 10 minutes of illumination. In a solution saturated with oxygen, the average bleaching time was of the order of 1 minute. No blinking was observed in the absorption signal. On the basis of bleaching steps, we obtained an average absorption cross section of 4 angstroms2 for a single chromophore.

Absorption and scattering microscopy of single metal nanoparticles

Several recently developed detection techniques opened studies of individual metal nanoparticles (1-100 nm in diameter) in the optical far field. Eliminating averaging over the broad size and shape distributions produced by even the best of current synthesis methods, these studies hold great promise for gaining a deeper insight into many of the properties of metal nanoparticles, notably electronic and vibrational relaxation. All methods are based on detection of a scattered wave emitted either by the particle itself, or by its close environment. Direct absorption and interference techniques rely on the particles scattering and have similar limits in signal-to-noise ratio. The photothermal method uses a photo-induced change in the refractive index of the environment as an additional step to scatter a wave with a different wavelength. This leads to a considerable improvement in signal-to-background ratio, and thus to a much higher sensitivity. We briefly discuss and compare these various techniques, review the new results they generated so far, and conclude on their great potential for nanoscience and for single-molecule labelling in biological assays and live cells.

Single metal nanoparticles: optical detection, spectroscopy and applications

Since the first report on the far-field optical detection of single metal nanoparticles in the late 1990s, the field has rapidly developed and new methods and concepts have been introduced. Eliminating averaging over the broad size, shape and crystallinity distributions produced by even the best of current synthesis methods, these techniques have proven extremely useful for gaining a deeper insight into many of the properties of metal nanoparticles. These techniques have already led to the first applications specifically directed at using single particles. In this review we describe far-field optical techniques (both linear and nonlinear) that have sufficient sensitivity to detect single metal particles. We further discuss emerging applications, and emphasize the importance of single-particle detection techniques in their development.

Photon bunching in the fluorescence from single molecules: A probe for intersystem crossing

Fluorescence photons emitted by a single molecule trapped in a solid at low temperature are correlated in time. A simple theory of the correlation pattern is presented, including in the same formalism photon antibunching from coherent Rabi oscillations and photon bunching due to incoherent quantum transitions between electronic levels. The correlation method is applied to single pentacene molecules in a para‐terphenyl crystal. A clear photon bunching allows us to determine the ISC rates for each molecule. We attribute the scatter of our results for the transition rate between excited singlet and triplet states, and the difference with the average value taken from the literature, to molecular distortions induced by crystal defects in our small sample. We conclude that the correlation method associated to single molecule spectroscopy has a great potential to study dynamical processes on intermediate time scales in condensed matter.

Resonant plasmonic enhancement of single-molecule fluorescence by individual gold nanorods

Enhancing the fluorescence of a weak emitter is important to further extend the reach of single-molecule fluorescence imaging to many unexplored systems. Here we study fluorescence enhancement by isolated gold nanorods and explore the role of the surface plasmon resonance (SPR) on the observed enhancements. Gold nanorods can be cheaply synthesized in large volumes, yet we find similar fluorescence enhancements as literature reports on lithographically fabricated nanoparticle assemblies. The fluorescence of a weak emitter, crystal violet, can be enhanced more than 1000-fold by a single nanorod with its SPR at 629 nm excited at 633 nm. This strong enhancement results from both an excitation rate enhancement of ∼130 and an effective emission enhancement of ∼9. The fluorescence enhancement, however, decreases sharply when the SPR wavelength moves away from the excitation laser wavelength or when the SPR has only a partial overlap with the emission spectrum of the fluorophore. The reported measurements of fluorescence enhancement by 11 nanorods with varying SPR wavelengths are consistent with numerical simulations.

Spectral diffusion and individual two-level systems probed by fluorescence of single terrylene molecules in a polyethylene matrix

Abstract We study the influence of low-energy matrix excitations on the line widths and fluorescence correlation of single terrylene molecules in polyethylene at helium temperatures. The histogram of line widths has a cutoff at the natural line width of terrylene, showing that for some molecules dephasing and spectral diffusion are negligible on the measurement time scale. The shape of the histogram can be qualitatively interpreted by means of a simple model for spectral diffusion. The line widths of different molecules show different temperature dependences. The correlation method is then applied to a time-resolved study of the intensity fluctuations of single molecule fluorescence. Many possible shapes of correlation functions appear, spanning many decades of relaxation times. We believe single two-level systems (TLSs) are the cause of the well defined exponential steps we observe. In some cases, the two positions of a single molecules line can be identified in the spectrum. They present the same time constant in their correlation functions and jump together to a new frequency. The dependence of the correlation on exciting flux shows that jumps can be spontaneous or photo-induced. The study of the rate as a function of temperature shows clear power laws which we attribute to tunnelling of the TLS assisted by one (T1) or two (T3) acoustic phonons. In one case, we find an Arrhenius activation of the rate, which could be explained by dressing the standard TLS model with slow matrix modes.

Single molecule lines and spectral hole burning of terrylene in different matrices

We observe fluorescence excitation lines of single terrylene molecules in three new polymer matrices (polyvinylbutyral, polymethylmethacrylate, and polystyrene) and in two crystals, n‐hexadecane (polycrystalline Shpol’skii matrix) and anthracene single crystal. We also burn persistent spectral holes in bulk samples of these solutions for comparison to single molecule lines. In all matrices where hole burning is efficient enough, we find good agreement between the average width determined from the distribution of single molecules’ linewidths and the homogeneous width deduced from spectral holes, which demonstrates the consistency and complementarity of the two techniques.

Luminescence Quantum Yield of Single Gold Nanorods

We study the luminescence quantum yield (QY) of single gold nanorods with different aspect ratios and volumes. Compared to gold nanospheres, we observe an increase of QY by about an order of magnitude for particles with a plasmon resonance >650 nm. The observed trend in QY is further confirmed by controlled reshaping of a single gold nanorod to a spherelike shape. Moreover, we identify two spectral components, one around 500 nm originating from a combination of interband transitions and the transverse plasmon and one coinciding with the longitudinal plasmon band. These components are analyzed by correlating scattering and luminescence spectra of single nanorods and performing polarization sensitive measurements. Our study contributes to the understanding of luminescence from gold nanorods. The enhanced QY we report can benefit applications in biological and soft matter studies.

Acoustic Oscillations and Elastic Moduli of Single Gold Nanorods

We present the first acoustic vibration measurements of single gold nanorods with well-characterized dimensions and crystal structure. The nanorods have an average size of 90 nm x 30 nm and display two vibration modes, the breathing mode and the extensional mode. Correlation between the dimensions obtained from electron microscope images and the vibrational frequencies of the same particle allows us to determine the elastic moduli for each individual nanorod. Contrary to previous reports on ensembles of gold nanorods, we find that the single particle elastic moduli agree well with bulk values.