Fisher information for smart sampling in time-domain spectroscopy.

Time-domain spectroscopy encompasses a wide range of techniques, such as Fourier-transform infrared, pump-probe, Fourier-transform Raman, and two-dimensional electronic spectroscopies. These methods enable various applications, such as molecule characterization, excited state dynamics studies, or spectral classification. Typically, these techniques rarely use sampling schemes that exploit the prior knowledge scientists typically have before the actual experiment. Indeed, not all sampling coordinates carry the same amount of information, and a careful selection of the sampling points may notably affect the resulting performance. In this work, we rationalize, with examples, the various advantages of using an optimal sampling scheme tailored to the specific experimental characteristics and/or expected results. We show that using a sampling scheme optimizing the Fisher information minimizes the variance of the desired parameters. This can greatly improve, for example, spectral classifications and multidimensional spectroscopy. We demonstrate how smart sampling may reduce the acquisition time of an experiment by one to two orders of magnitude, while still providing a similar level of information.

One-Shot Phase Image Distinction of Plasmonic and Dielectric Nanoparticles.

Nanoscale phase control is one of the most powerful approaches to specifically tailor electrical fields in modern nanophotonics. Especially the precise subwavelength assembly of many individual nanobuilding blocks has given rise to exciting new materials as diverse as metamaterials, for miniaturizing optics, or 3D assembled plasmonic structures for biosensing applications. Despite its fundamental importance, the phase response of individual nanostructures is experimentally extremely challenging to visualize. Here, we address this shortcoming and measure the quantitative scattering phase of different nanomaterials such as gold nanorods and spheres as well as dielectric nanoparticles. Beyond reporting spectrally resolved responses, with phase changes close to π when passing the particles' plasmon resonance, we devise a simple method for distinguishing different plasmonic and dielectric particles purely based on their phase behavior. Finally, we integrate this novel approach in a single-shot two-color scheme, capable of directly identifying different types of nanoparticles on one sample, from a single widefield image.

Precise Nanosizing with High Dynamic Range Holography.

Optical sensing is one of the key enablers of modern diagnostics. Especially label-free imaging modalities hold great promise as they eliminate labeling procedures prior to analysis. However, scattering signals of nanometric particles scale with their volume square. This unfavorable scaling makes it extremely difficult to quantitatively characterize intrinsically heterogeneous clinical samples, such as extracellular vesicles, as their signal variation easily exceeds the dynamic range of currently available cameras. Here, we introduce off-axis k-space holography that circumvents this limitation. By imaging the back-focal plane of our microscope, we project the scattering signal of all particles onto all camera pixels, thus dramatically boosting the achievable dynamic range to up to 110 dB. We validate our platform by detecting and quantitatively sizing metallic and dielectric particles over a 200 × 200 µm field of view and demonstrate that independently performed signal calibrations allow correctly sizing particles made from different materials. Finally, we present quantitative size distributions of extracellular vesicle samples.

Ultrafast Transient Holographic Microscopy.

Nanotechnology is increasingly being applied in many emerging technologies, ranging from metamaterials to next-generation nanodrugs. A key ingredient for its success is the ability to specifically tailor ultrafast nanoscale light-matter interactions over very large areas. Unfortunately, dynamic imaging by ultrafast nanoscopy so far remains limited to very small 2D areas. This shortcoming prevents connecting single-particle observations with large-scale functionality. Here, we address this experimental challenge by combining concepts of ultrafast spectroscopy, wide-field nanoscopy, and digital holography. We introduce an ultrafast holographic transient microscope for wide-field transient nanoscale imaging with high frequency all-optical signal demodulation. We simultaneously record ultrafast transient dynamics of many individual nano-objects and demonstrate time-resolved spectroscopy of gold nanoparticles over a large volume irrespective of their x-y-z position. Our results pave the way to single-shot 3D microscopy of 2D and 3D materials on arbitrary time scales from femtosecond carrier dynamics in optoelectronic materials to millisecond dynamics in complex tissues.

Widefield SERS for High-Throughput Nanoparticle Screening.

Surface-enhanced Raman scattering (SERS) imaging is a powerful technology with unprecedent potential for ultrasensitive chemical analysis. Point-by-point scanning and often excessively long spectral acquisition-times hamper the broad exploitation of the full analytical potential of SERS. Here, we introduce large-scale SERS particle screening (LSSPS), a multiplexed widefield screening approach to particle characterization, which is 500-1000 times faster than typical confocal Raman implementations. Beyond its higher throughput, LSSPS simultaneously quantifies both the sample's Raman and Rayleigh scattering to directly quantify the fraction of SERS-active particles which allows for an unprecedented correlation of SERS activity with particle size. .

Control of Vibronic Transition Rates by Resonant Single-Molecule-Nanoantenna Coupling.

Plasmonic nanostructures dramatically alter the radiative and nonradiative properties of single molecules in their vicinity. This coupling-induced change in decay channels selectively enhances specific vibronic transitions, which can enable plasmonic control of molecular reactivity. Here, we report coupling-dependent spectral emission shaping of single Rhodamine 800 molecules in the vicinity of plasmonic gold nanorods. We show that the relative vibronic transition rates of the first two vibronic transitions of the spontaneous emission spectrum can be tuned in the weak coupling regime, by approximately 25-fold, on the single molecule level.

Cold and Hot Spots: From Inhibition to Enhancement by Nanoscale Phase Tuning of Optical Nanoantennas.

Optical nanoantennas are well-known for the confinement of light into nanoscale hot spots, suitable for emission enhancement and sensing applications. Here, we show how control of the antenna dimensions allows tuning the local optical phase, hence turning a hot spot into a cold spot. We manipulate the local intensity exploiting the interference between driving and scattered field. Using single molecules as local detectors, we experimentally show the creation of subwavelength pockets with full suppression of the driving field. Remarkably, together with the cold excitation spots, we observe inhibition of emission by the phase-tuned nanoantenna. The fluorescence lifetime of a molecule scanned in such volumes becomes longer, showing slow down of spontaneous decay. In conclusion, the spatial phase of a nanoantenna is a powerful knob to tune between enhancement and inhibition in a 3-dimensional subwavelength volume.

Nanoscale Imaging and Control of Hexagonal Boron Nitride Single Photon Emitters by a Resonant Nanoantenna.

Defect centers in two-dimensional hexagonal boron nitride (hBN) are drawing attention as single-photon emitters with high photostability at room temperature. With their ultrahigh photon-stability, hBN single-photon emitters are promising for new applications in quantum technologies and for 2D-material based optoelectronics. Here, we control the emission rate of hBN-defects by coupling to resonant plasmonic nanocavities. By deterministic control of the antenna, we acquire high-resolution emission maps of the single hBN-defects. Using time-gating, we can discriminate the hBN-defect emission from the antenna luminescence. We observe sharp dips (40 nm fwhm) in emission, together with a reduction in luminescence lifetime. Comparing with finite-difference time-domain simulations, we conclude that both radiative and nonradiative rates are enhanced, effectively reducing the quantum efficiency. Also, the large refractive index of hBN largely screens off the local antenna field enhancement. Finally, based on the insight gained we propose a close-contact design for an order of magnitude brighter hBN single-photon emission.

Nanoscale Mapping and Control of Antenna-Coupling Strength for Bright Single Photon Sources.

Cavity quantum electrodynamics is the art of enhancing light-matter interaction of photon emitters in cavities with opportunities for sensing, quantum information, and energy capture technologies. To boost emitter-cavity interaction, that is, coupling strength g, ultrahigh quality cavities have been concocted yielding photon trapping times of microsecondsy to milliseconds. However, such high- Q cavities give poor photon output, hindering applications. To preserve high photon output, it is advantageous to strive for highly localized electric fields in radiatively lossy cavities. Nanophotonic antennas are ideal candidates combining low- Q factors with deeply localized mode volumes, allowing large g, provided the emitter is positioned exactly right inside the nanoscale mode volume. Here, with nanometer resolution, we map and tune the coupling strength between a dipole nanoantenna-cavity and a single molecule, obtaining a coupling rate of gmax ∼ 200 GHz. Together with accelerated single photon output, this provides ideal conditions for fast and pure nonclassical single photon emission with brightness exceeding 109 photons/sec. Clearly, nanoantennas acting as "bad" cavities offer an optimal regime for strong coupling g to deliver bright on-demand and ultrafast single photon nanosources for quantum technologies.

Enhancing Magnetic Light Emission with All-Dielectric Optical Nanoantennas.

Electric and magnetic optical fields carry the same amount of energy. Nevertheless, the efficiency with which matter interacts with electric optical fields is commonly accepted to be at least 4 orders of magnitude higher than with magnetic optical fields. Here, we experimentally demonstrate that properly designed photonic nanoantennas can selectively manipulate the magnetic versus electric emission of luminescent nanocrystals. In particular, we show selective enhancement of magnetic emission from trivalent europium-doped nanoparticles in the vicinity of a nanoantenna tailored to exhibit a magnetic resonance. Specifically, by controlling the spatial coupling between emitters and an individual nanoresonator located at the edge of a near-field optical scanning tip, we record with nanoscale precision local distributions of both magnetic and electric radiative local densities of states (LDOS). The map of the radiative LDOS reveals the modification of both the magnetic and electric quantum environments induced by the presence of the nanoantenna. This manipulation and enhancement of magnetic light-matter interaction by means of nanoantennas opens up new possibilities for the research fields of optoelectronics, chiral optics, nonlinear and nano-optics, spintronics, and metamaterials, among others.

Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins.

Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.

In-Plane Plasmonic Antenna Arrays with Surface Nanogaps for Giant Fluorescence Enhancement.

Optical nanoantennas have a great potential for enhancing light-matter interactions at the nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate antenna performance and applications. In most designs, the region of maximum field localization and enhancement (i.e., hotspot) is not readily accessible to the sample because it is buried into the nanostructure. Moreover, current large-scale fabrication techniques lack reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication technique that applies planarization, etch back, and template stripping to expose the excitation hotspot at the surface, providing a major improvement over conventional electron beam lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface accessibility of the hotspot confined region. The novel fabrication approach drastically improves the optical performance of plasmonic nanoantennas to yield giant fluorescence enhancement factors up to 104-105 times, together with nanoscale detection volumes in the 20 zL range. The method is fully scalable and adaptable to a wide range of antenna designs. We foresee broad applications by the use of these in-plane antenna geometries ranging from large-scale ultrasensitive sensor chips to microfluidics and live cell membrane investigations.

Plasmonic Nanoantennas Enable Forbidden Förster Dipole-Dipole Energy Transfer and Enhance the FRET Efficiency.

Förster resonance energy transfer (FRET) plays a key role in biochemistry, organic photovoltaics, and lighting sources. FRET is commonly used as a nanoruler for the short (nanometer) distance between donor and acceptor dyes, yet FRET is equally sensitive to the mutual dipole orientation. The orientation dependence complicates the FRET analysis in biological samples and may even lead to the absence of FRET for perpendicularly oriented donor and acceptor dipoles. Here, we exploit the strongly inhomogeneous and localized fields in plasmonic nanoantennas to open new energy transfer routes, overcoming the limitations from the mutual dipole orientation to ultimately enhance the FRET efficiency. We demonstrate that the simultaneous presence of perpendicular near-field components in the nanoantenna sets favorable energy transfer routes that increase the FRET efficiency up to 50% for nearly perpendicular donor and acceptor dipoles. This new facet of plasmonic nanoantennas enables dipole-dipole energy transfer that would otherwise be forbidden in a homogeneous environment. As such, our approach further increases the applicability of single-molecule FRET over diffraction-limited approaches, with the additional benefits of higher sensitivities and higher concentration ranges toward physiological levels.

Visualizing and controlling vibrational wave packets of single molecules.

The active steering of the pathways taken by chemical reactions and the optimization of energy conversion processes provide striking examples of the coherent control of quantum interference through the use of shaped laser pulses. Experimentally, coherence is usually established by synchronizing a subset of molecules in an ensemble with ultra-short laser pulses. But in complex systems where even chemically identical molecules exist with different conformations and in diverse environments, the synchronized subset will have an intrinsic inhomogeneity that limits the degree of coherent control that can be achieved. A natural-and, indeed, the ultimate-solution to overcoming intrinsic inhomogeneities is the investigation of the behaviour of one molecule at a time. The single-molecule approach has provided useful insights into phenomena as diverse as biomolecular interactions, cellular processes and the dynamics of supercooled liquids and conjugated polymers. Coherent state preparation of single molecules has so far been restricted to cryogenic conditions, whereas at room temperature only incoherent vibrational relaxation pathways have been probed. Here we report the observation and manipulation of vibrational wave-packet interference in individual molecules at ambient conditions. We show that adapting the time and phase distribution of the optical excitation field to the dynamics of each molecule results in a high degree of control, and expect that the approach can be extended to achieve single-molecule coherent control in other complex inhomogeneous systems.

Plasmonic antennas as design elements for coherent ultrafast nanophotonics.

Broadband excitation of plasmons allows control of light-matter interaction with nanometric precision at femtosecond timescales. Research in the field has spiked in the past decade in an effort to turn ultrafast plasmonics into a diagnostic, microscopy, computational, and engineering tool for this novel nanometric-femtosecond regime. Despite great developments, this goal has yet to materialize. Previous work failed to provide the ability to engineer and control the ultrafast response of a plasmonic system at will, needed to fully realize the potential of ultrafast nanophotonics in physical, biological, and chemical applications. Here, we perform systematic measurements of the coherent response of plasmonic nanoantennas at femtosecond timescales and use them as building blocks in ultrafast plasmonic structures. We determine the coherent response of individual nanoantennas to femtosecond excitation. By mixing localized resonances of characterized antennas, we design coupled plasmonic structures to achieve well-defined ultrafast and phase-stable field dynamics in a predetermined nanoscale hotspot. We present two examples of the application of such structures: control of the spectral amplitude and phase of a pulse in the near field, and ultrafast switching of mutually coherent hotspots. This simple, reproducible and scalable approach transforms ultrafast plasmonics into a straightforward tool for use in fields as diverse as room temperature quantum optics, nanoscale solid-state physics, and quantum biology.

Matching Nanoantenna Field Confinement to FRET Distances Enhances Förster Energy Transfer Rates.

Förster resonance energy transfer (FRET) is widely applied in chemistry, biology, and nanosciences to assess distances on sub-10 nm scale. Extending the range and applicability of FRET requires enhancement of the fluorescence energy transfer at a spatial scale comparable to the donor-acceptor distances. Plasmonic nanoantennas are ideal to concentrate optical fields at a nanoscale fully matching the FRET distance range. Here, we present a resonant aluminum nanogap antenna tailored to enhance single molecule FRET. A 20 nm gap confines light into a nanoscale volume, providing a field gradient on the scale of the donor-acceptor distance, a large 10-fold increase in the local density of optical states, and strong intensity enhancement. With our dedicated design, we obtain 20-fold enhancement on the fluorescence emission of donor and acceptor dyes, and most importantly up to 5-fold enhancement of the FRET rate for donor-acceptor separations of 10 nm. We also provide a thorough framework of the fluorescence photophysics occurring in the nanoscale gap volume. The presented enhancement of energy transfer flow at the nanoscale opens a yet unexplored facet of the various advantages of optical nanoantennas and provides a new strategy toward biological applications of single molecule FRET at micromolar concentrations.

Single molecule microscopy and spectroscopy: concluding remarks.

Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what "Single Molecule Detection" provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on "Single Molecule Microscopy and Spectroscopy" was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (). Surely, with the questioning eyes of his bust on the 1st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives.

Multicolour single molecule emission and excitation spectroscopy reveals extensive spectral shifts.

We explore the distribution and shape of single molecule spectra at room temperature, when embedded in a polymer host. Multicolour excitation and emission spectroscopy is implemented to capture the full inhomogeneous distribution. We observe dramatic spectral changes in a distribution of single quaterrylene diimide (QDI) molecules isolated in a PMMA matrix. The molecules are strongly blue shifted with respect to the ensemble absorption maximum and spread over a staggering 200 nm range. Despite these strong shifts, the shape of the emission spectra does not differ much between individual molecules. We demonstrate that a considerable number of molecules may be invisible in single molecule experiments, as they typically rely on only a single excitation wavelength, which predetermines which subensemble is probed in the experiment. Lastly, we make a first step towards single molecule excitation spectroscopy under ambient conditions, which allows us to determine the spectral range at which individual molecules absorb light most efficiently. We show how single molecule emission and excitation spectroscopies can complement each other and a combination of both techniques can help in understanding the origin of underlaying spectral properties of individual molecules.

Ultrafast dynamics of single molecules.

The detection of individual molecules has found widespread application in molecular biology, photochemistry, polymer chemistry, quantum optics and super-resolution microscopy. Tracking of an individual molecule in time has allowed identifying discrete molecular photodynamic steps, action of molecular motors, protein folding, diffusion, etc. down to the picosecond level. However, methods to study the ultrafast electronic and vibrational molecular dynamics at the level of individual molecules have emerged only recently. In this review we present several examples of femtosecond single molecule spectroscopy. Starting with basic pump-probe spectroscopy in a confocal detection scheme, we move towards deterministic coherent control approaches using pulse shapers and ultra-broad band laser systems. We present the detection of both electronic and vibrational femtosecond dynamics of individual fluorophores at room temperature, showing electronic (de)coherence, vibrational wavepacket interference and quantum control. Finally, two colour phase shaping applied to photosynthetic light-harvesting complexes is presented, which allows investigation of the persistent coherence in photosynthetic complexes under physiological conditions at the level of individual complexes.

Vectorial nanoscale mapping of optical antenna fields by single molecule dipoles.

Optical nanoantennas confine light on the nanoscale, enabling strong light-matter interactions and ultracompact optical devices. Such confined nanovolumes of light have nonzero field components in all directions (x, y, and z). Unfortunately mapping of the actual nanoscale field vectors has so far remained elusive, though antenna hotspots have been explored by several techniques. In this paper, we present a novel method to probe all three components of the local antenna field. To this end a resonant nanoantenna is fabricated at the vertex of a scanning tip. Next, the nanoantenna is deterministically scanned in close proximity to single fluorescent molecules, whose fixed excitation dipole moment reads out the local field vector. With nanometer molecular resolution, we distinctly map x-, y-, and z-field components of the dipole antenna, i.e. a full vectorial mode map, and show good agreement with full 3D FDTD simulations. Moreover, the fluorescence polarization maps the localized coupling, with emission through the longitudinal antenna mode. Finally, the resonant antenna probe is used for single molecule imaging with 40 nm fwhm response function. The total fluorescence enhancement is 7.6 times, while out-of-plane molecules, almost undetectable in far-field, are made visible by the strong antenna z-field with a fluorescence enhancement up to 100 times. Interestingly, the apparent position of molecules shifts up to 20 nm depending on their orientation. The capability to resolve orientational information on the single molecule level makes the scanning resonant antenna an ideal tool for extreme resolution bioimaging.