Single-Particle Photothermal Circular Dichroism and Photothermal Magnetic Circular Dichroism Microscopy.

Recent advances in single-particle photothermal circular dichroism (PT CD) and photothermal magnetic circular dichroism (PT MCD) microscopy have shown strong promise for diverse applications in chirality and magnetism. Photothermal circular dichroism microscopy measures direct differential absorption of left- and right-circularly polarized light by a chiral nanoobject and thus can measure a pure circular dichroism signal, which is free from the contribution of circular birefringence and linear dichroism. Photothermal magnetic circular dichroism, which is based on the polar magneto-optical Kerr effect, can probe the magnetic properties of a single nanoparticle (of sizes down to 20 nm) optically. Single-particle measurements enable studies of the spatiotemporal heterogeneity of magnetism at the nanoscale. Both PT CD and PT MCD have already found applications in chiral plasmonics and magnetic nanomaterials. Most importantly, the advent of these microscopic techniques opens possibilities for many novel applications in biology and nanomaterial science.

Exploring Rotational Diffusion with Plasmonic Coupling.

Measuring the orientation dynamics of nanoparticles and nonfluorescent molecules in real time with optical methods is still a challenge in nanoscience and biochemistry. Here, we examine optoplasmonic sensing taking the rotational diffusion of plasmonic nanorods as an experimental model. Our detection method is based on monitoring the dark-field scattering of a relatively large sensor gold nanorod (GNR) (40 nm in diameter and 112 nm in length) as smaller plasmonic nanorods cross its near field. We observe the rotational motion of single small gold nanorods (three samples with about 5 nm in diameter and 15.5, 19.1, and 24.6 nm in length) in real time with a time resolution around 50 ns. Plasmonic coupling enhances the signal of the diffusing gold nanorods, which are 1 order of magnitude smaller in volume (about 300 nm3) than those used in our previous rotational diffusion experiments. We find a better angular sensitivity with plasmonic coupling in comparison to the free diffusion in the confocal volume. Yet, the angle sensitivity we find with plasmonic coupling is reduced compared to the sensitivity expected from simulations at fixed positions due to the simultaneous translational and rotational diffusion of the small nanorods. To get a reliable plasmonic sensor with the full angular sensitivity, it will be necessary to construct a plasmonic assembly with positions and orientations nearly fixed around the optimum geometry.

Future Paths in Cryogenic Single-Molecule Fluorescence Spectroscopy.

In the last three decades, cryogenic single-molecule fluorescence spectroscopy has provided average-free understanding of the photophysics and of fundamental interactions at molecular scales. Furthermore, they propose original pathways and applications in the treatment and storage of quantum information. The ultranarrow lifetime-limited zero-phonon line acts as an excellent sensor to local perturbations caused either by intrinsic dynamical degrees of freedom, or by external perturbations, such as those caused by electric fields, elastic and acoustic deformations, or light-induced dynamics. Single aromatic hydrocarbon molecules, being sensitive to nanoscale probing at nanometer scales, are potential miniaturized platforms for integrated quantum photonics. In this Perspective, we look back at some of the past advances in cryogenic optical microscopy and propose some perspectives for future development.

Probing the in-plane dipole moment vector between ground and excited state of single molecules by the Stark effect.

Single molecules, embedded inside a well-defined insertion site of a single-crystalline host matrix, are sensitive probes of electric field via the induced Stark shift on their lifetime-limited electronic transition. Though the response of molecules to electric field has been shown to be relatively homogeneous, crystal symmetry allows for several, spectroscopically-indistinguishable, orientations of the net permanent dipole moment between the ground and excited state - the dipole vector - and this is problematic for measuring field orientation and magnitude. In this work, we measure for each terrylene molecule, embedded inside a new host matrix, the dipole vector independently by an electric field that we can rotate in the plane of the crystal. This single crystal host matrix, called [1]BenzoThieno[3,2-b]BenzoThiophene, induces a moderate symmetry breaking of the embedded centrosymmetric terrylene molecule, and gives rise to a net dipole moment of 0.28±0.09 Debye. Based on quantum chemistry calculations we propose an insertion site that best matches the experimental findings.

Sharp zero-phonon lines of single organic molecules on a hexagonal boron-nitride surface.

Single fluorescent molecules embedded in the bulk of host crystals have proven to be sensitive probes of the dynamics in their nano environment, thanks to their narrow (about 30-50 MHz or 0.1-0.2 µeV) optical linewidth of the 0-0 zero-phonon line (0-0 ZPL) at cryogenic temperatures. However, the optical linewidths of the 0-0 ZPL have been found to increase dramatically as the single molecules are located closer to a surface or interface, while no 0-0 ZPL has been detected for single molecules on any surface. Here we study single terrylene molecules adsorbed on the surface of hexagonal boron-nitride (hBN) substrates. Our low-temperature results show that it is possible to observe the 0-0 ZPL of fluorescent molecules on a surface. We compare our results for molecules deposited on the surfaces of annealed and non-annealed hBN flakes and we see a marked improvement in the spectral stability of the emitters after annealing.

Burst-by-Burst Measurement of Rotational Diffusion at Nanosecond Resolution Reveals Hot-Brownian Motion and Single-Chain Binding.

We record dark-field scattering bursts of individual gold nanorods, 52 × 15 nm2 in average size, freely diffusing in water suspension. We deduce their Brownian rotational diffusion constant from autocorrelation functions on a single-event basis. Due to spectral selection by the plasmonic resonance with the excitation laser, the distribution of rotational diffusion constants is much narrower than expected from the size distribution measured by TEM. As rotational diffusion depends on particle hydrodynamic volume, viscosity, and temperature, it can sense those parameters at the single-particle level. We demonstrate measurements of hot Brownian rotational diffusion of nanorods in temperature and viscosity gradients caused by plasmonic heating. Further, we monitor hydrodynamic volumes of gold nanorods upon addition of very low concentrations of the water-soluble polymer PVA, which binds to the particles, leading to measurable changes in their diffusion constant corresponding to binding of one to a few polymer coils. We propose this analysis technique for very low concentrations of biomolecules in solution.

Dynamic Heterogeneity in the Optical Signals from Single Nano-Objects.

In contrast to ensemble-averaged measurements, single-molecule experiments directly display the heterogeneity of molecular properties in space and time. In many complex systems, spatial heterogeneity is regularly accompanied by temporal or dynamic heterogeneity; if a property differs from molecule to molecule, it will often vary in time for one and the same molecule. In this short paper, we discuss a few examples of complex systems where dynamical heterogeneity was observed in single-molecule or single-particle optical signals. For single biomolecules, the first demonstration of dynamic heterogeneity in a single enzyme was provided by Xie and colleagues. Other examples are found in glassy systems, and very recently in the magnetic relaxation of single superparamagnetic nanoparticles. The ubiquity of this phenomenon suggests that, rather than an exception, dynamic heterogeneity is the rule in complex systems with multiple degrees of freedom.

Thousand-Fold Enhancement of Photothermal Signals in Near-Critical CO2.

Photothermal (PT) microscopy has shown strong promise in imaging single absorbing nano-objects in soft matter and biological systems. PT imaging at ambient conditions usually requires a high laser power for a sensitive detection, which prevents application to light-sensitive nanoparticles. In a previous study of single gold nanoparticles, we showed that the photothermal signal can be enhanced more than 1000-fold in near-critical xenon compared to that in glycerol, a typical medium for PT detection. In this report, we show that carbon dioxide (CO2), a much cheaper gas than xenon, can enhance PT signals in a similar way. We confine near-critical CO2 in a thin capillary which easily withstands the high near-critical pressure (around 74 bar) and facilitates sample preparation. We also demonstrate enhancement of the magnetic circular dichroism signal of single magnetite nanoparticle clusters in supercritical CO2. We have performed COMSOL simulations to support and explain our experimental findings.

Photothermal Circular Dichroism Measurements of Single Chiral Gold Nanoparticles Correlated with Electron Tomography.

Chemically synthesized metal nanoparticles with morphological chiral features are known to exhibit strong circular dichroism. However, we still lack understanding of the correlation between morphological and chiroptical features of plasmonic nanoparticles. To shed light on that question, single nanoparticle experiments are required. We performed photothermal circular dichroism measurements of single chiral and achiral gold nanoparticles and correlated the chiroptical response to the 3D morphology of the same nanoparticles retrieved by electron tomography. In contrast to an ensemble measurement, we show that individual particles within the ensemble display a broad distribution of strength and handedness of circular dichroism signals. Whereas obvious structural chiral features, such as helical wrinkles, translate into chiroptical ones, nanoparticles with less obvious chiral morphological features can also display strong circular dichroism signals. Interestingly, we find that even seemingly achiral nanoparticles can display large g-factors. The origin of this circular dichroism signal is discussed in terms of plasmonics and other potentially relevant factors.

Optically Probing the Chirality of Single Plasmonic Nanostructures and of Single Molecules: Potential and Obstacles.

Circular dichroism (CD) is a standard method for the analysis of biomolecular conformation. It is very reliable when applied to molecules, but requires relatively large amounts of solution. Plasmonics offer the perspective of enhancement of CD signals, which would extend CD spectrometry to smaller amounts of molecules and to weaker chiral signals. However, plasmonic enhancement comes at the cost of additional complications: averaging over all orientations is no longer possible or reliable, linear dichroism leaks into CD signals because of experimental imperfections, scattering and its interference with the incident beam must be taken into account, and the interaction between chiral molecules and possibly chiral plasmonic structures considerably complicates the interpretation of measured signals. This Perspective aims to explore the motivations and problems of plasmonic chirality and to re-evaluate present and future solutions.

Ultrasensitive detection of local acoustic vibrations at room temperature by plasmon-enhanced single-molecule fluorescence.

Sensitive detection of local acoustic vibrations at the nanometer scale has promising potential applications involving miniaturized devices in many areas, such as geological exploration, military reconnaissance, and ultrasound imaging. However, sensitive detection of weak acoustic signals with high spatial resolution at room temperature has become a major challenge. Here, we report a nanometer-scale system for acoustic detection with a single molecule as a probe based on minute variations of its distance to the surface of a plasmonic gold nanorod. This system can extract the frequency and amplitude of acoustic vibrations with experimental and theoretical sensitivities of 10 pm Hz-1/2 and 10 fm Hz-1/2, respectively. This approach provides a strategy for the optical detection of acoustic waves based on molecular spectroscopy without electromagnetic interference. Moreover, such a small nano-acoustic detector with 40-nm size can be employed to monitor acoustic vibrations or read out the quantum states of nanomechanical devices.

Two-Photon-Excited Single-Molecule Fluorescence Enhanced by Gold Nanorod Dimers.

We demonstrate two-photon-excited single-molecule fluorescence enhancement by single end-to-end self-assembled gold nanorod dimers. We employed biotinylated streptavidin as the molecular linker, which connected two gold nanorods in end-to-end fashion. The typical size of streptavidin of around 5 nm separates the gold nanorods with gaps suitable for the access of fresh dyes in aqueous solution, yet small enough to give very high two-photon fluorescence enhancement. Simulations show that enhancements of more than 7 orders of magnitude can be achieved for two-photon-excited fluorescence in the plasmonic hot spots. With such high enhancements, we successfully detect two-photon-excited fluorescence for a common organic dye (ATTO 610) at the single-molecule, single-nanoparticle level.

Controlled synthesis of gold nanorod dimers with end-to-end configurations.

End-to-end gold nanorod dimers provide unique plasmonic hotspots with extremely large near-field enhancements in the gaps. Thereby they are beneficial in a wide range of applications, such as enhancing the emissions from ultra-weak emitters. For practical purposes, synthesis of gold nanorod dimers with high yield, especially on the substrates, is essential. Here, we demonstrate two controllable strategies to synthesize gold nanorod dimers based on the self-assembly of gold nanorods, either in bulk solution or on the surface of a glass substrate directly. Both methods can give a high yield of gold nanorod dimers, yet, assembling them directly on the substrate provides more flexibility in controlling the shape and size of each nanorod within the dimer. We also show that these gold nanorod dimers can be used to enhance two-photon-excited fluorescence signals at the single-molecule level.

Progress and perspectives in single-molecule optical spectroscopy.

We review some of the progress of single-molecule optical experiments in the past 20 years and propose some perspectives for the coming years. We particularly focus on methodological advances in fluorescence, super-resolution, photothermal contrast, and interferometric scattering and briefly discuss a few of the applications. These advances have enabled the exploration of new emitters and quantum optics; the chemistry and biology of complex heterogeneous systems, nanoparticles, and plasmonics; and the detection and study of non-fluorescing and non-absorbing nano-objects. We conclude by proposing some ideas for future experiments. The field will move toward more and better signals of a broader variety of objects and toward a sharper view of the surprising complexity of the nanoscale world of single (bio-)molecules, nanoparticles, and their nano-environments.

Imaging the Magnetization of Single Magnetite Nanoparticle Clusters via Photothermal Circular Dichroism.

Magnetic imaging is a versatile tool in biological and condensed-matter physics. Existing magnetic imaging techniques either require demanding experimental conditions which restrict the range of their applications or lack the spatial resolution required for single-particle measurements. Here, we combine photothermal (PT) microscopy with magnetic circular dichroism (MCD) to develop a versatile magnetic imaging technique using visible light. Unlike most magnetic imaging techniques, photothermal magnetic circular dichroism (PT MCD) microscopy works particularly well for single nanoparticles immersed in liquids. As a proof of principle, we demonstrate magnetic CD imaging of superparamagnetic magnetite nanoparticulate clusters immersed in microscope immersion oil. The sensitivity of our method allowed us to probe the magnetization curve of single ∼400-nm-diameter magnetite nanoparticulate clusters.

Reverse Intersystem Crossing of Single Deuterated Perylene Molecules in a Dibenzothiophene Matrix.

The front cover artwork is provided by Prof. Michel Orrit's group at the University of Leiden, The Netherlands. The image shows the structures of the dibenzothiophene host molecule and perylene guest molecule with its fluorescence emission spectrum on the bottom. The symbols and arrows refer to the reverse intersystem crossing (rISC) observed for single perylene molecules in dibenzothiophene host crystals, which typically have a needle shape and are shown in the background. Read the full text of the Article at 10.1002/cphc.202100679.

Nanosecond time scale transient optoplasmonic detection of single proteins.

Optical detection of individual proteins with high bandwidth holds great promise for understanding important biological processes on the nanoscale and for high-throughput fingerprinting applications. As fluorescent labels impose restrictions on detection bandwidth and require time-intensive and invasive processes, label-free optical techniques are highly desirable. Here, we read out changes in the resonantly scattered field of individual gold nanorods interferometrically and use photothermal spectroscopy to optimize the experiment's parameters. This interferometric plasmonic scattering enables the observation of single proteins as they traverse plasmonic near fields of gold nanorods with unprecedented temporal resolution in the nanosecond-to-microsecond range.

Reverse Intersystem Crossing of Single Deuterated Perylene Molecules in a Dibenzothiophene Matrix.

Intersystem crossing to the long-lived metastable triplet state is often a strong limitation on fluorescence brightness of single molecules, particularly for perylene in various matrices. In this paper, we report on a strong excitation-induced reverse intersystem crossing (rISC), a process where single perylene molecules in a dibenzothiophene matrix recover faster from the triplet state, turning into bright emitters at saturated excitation powers. With a detailed study of single-molecule fluorescence autocorrelations, we quantify the effect of rISC. The intrinsic lifetimes found for the two effective triplet states (8.5±0.4 ms and 64±12 ms) become significantly shorter, into the sub-millisecond range, as the excitation power increases and fluorescence brightness is ultimately enhanced at least fourfold. Our results are relevant for the understanding of triplet state manipulation of single-molecule quantum emitters and for markedly improving their brightness.

Photothermal Spectro-Microscopy as Benchmark for Optoplasmonic Bio-Detection Assays.

Optoplasmonic bio-detection assays commonly probe the response of plasmonic nanostructures to changes in their dielectric environment. The accurate detection of nanoscale entities such as virus particles, micelles and proteins requires optimization of multiple experimental parameters. Performing such optimization directly via analyte recognition is often not desirable or feasible, especially if the nanostructures exhibit limited numbers of analyte binding sites and if binding is irreversible. Here we introduce photothermal spectro-microscopy as a benchmarking tool for the characterization and optimization of optoplasmonic detection assays.

Photothermal Circular Dichroism of Single Nanoparticles Rejecting Linear Dichroism by Dual Modulation.

Circular dichroism (CD) is the property of chiral nanoobjects to absorb circularly polarized light of either handedness to different extents. Photothermal microscopy enables the detection of CD signals with high sensitivity and provides a direct absorptive response of the samples under study. To achieve CD measurements at the single-particle level, one must reduce such artifacts as leakage of linear dichroism (LD) and residual intensity modulation. We have simulated our setup with a simple model, which allows us to tune modulation parameters to obtain a CD signal virtually free from artifacts. We demonstrate the sensitivity of our setup by measuring the very weak inherent CD signals of single gold nanospheres. We furthermore demonstrate that our method can be extended to obtain spectra of the full absorptive properties of single nanoparticles, including isotropic absorption, linear dichroism, and circular dichroism. We then investigate nominally achiral gold nanoparticles immersed in a chiral liquid. Carefully taking into account the intrinsic chirality of the particles and its change due to heat-induced reshaping, we find that the chiral liquid carvone surrounding the particle has no measurable effect on the particles' chirality, down to g-factors of 3 × 10-4.