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.

FRET analysis of CP12 structural interplay by GAPDH and PRK.

CP12 is an intrinsically disordered protein playing a key role in the regulation of the Benson-Calvin cycle. Due to the high intrinsic flexibility of CP12, it is essential to consider its structural modulation induced upon binding to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) enzymes. Here, we report for the first time detailed structural modulation about the wild-type CP12 and its site-specific N-terminal and C-terminal disulfide bridge mutants upon interaction with GAPDH and PRK by Förster resonance energy transfer (FRET). Our results indicate an increase in CP12 compactness when the complex is formed with GAPDH or PRK. In addition, the distributions in FRET histograms show the elasticity and conformational flexibility of CP12 in all supra molecular complexes. Contrarily to previous beliefs, our FRET results importantly reveal that both N-terminal and C-terminal site-specific CP12 mutants are able to form the monomeric (GAPDH-CP12-PRK) complex.

FRET enhancement in aluminum zero-mode waveguides.

Zero-mode waveguides (ZMWs) can confine light into attoliter volumes, which enables single molecule fluorescence experiments at physiological micromolar concentrations. Of the fluorescence spectroscopy techniques that can be enhanced by ZMWs, Förster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentrations with single-molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs that diffuse into aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large amount of literature that describe their use for single-molecule fluorescence spectroscopy. We also compared the results between ZMWs milled in gold and aluminum, and found that although gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observed that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states. Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single-molecule FRET at physiological concentrations.

Nanophotonic enhancement of the Förster resonance energy-transfer rate with single nanoapertures.

Tailoring the light-matter interaction and the local density of optical states (LDOS) with nanophotonics provides accurate control over the luminescence properties of a single quantum emitter. This paradigm is also highly attractive to enhance the near-field Förster resonance energy transfer (FRET) between two fluorescent emitters. Despite the wide applications of FRET in nanosciences, using nanophotonics to enhance FRET has remained a debated and complex challenge. Here we demonstrate enhanced energy transfer within single donor-acceptor fluorophore pairs confined in single gold nanoapertures. Experiments monitoring both the donor and the acceptor emission photodynamics clearly establish a linear dependence of the FRET rate on the LDOS in nanoapertures, demonstrating that nanophotonics can be used to intensify the near-field energy transfer. Strikingly, we observe a significant six-fold increase in the FRET rate for large donor-acceptor separations exceeding 13 nm. Exciting opportunities are opened to investigate biochemical structures with donor-acceptor distances much beyond the classical Förster radius. Importantly, our approach is fully compatible with the detection of single biomolecules freely diffusing in water solution under physiological conditions.

Hollow-core photonic crystal fiber probe for remote fluorescence sensing with single molecule sensitivity.

Current optical fiber probes for fluorescence spectroscopy struggle with large luminescence background and low detection sensitivities that challenge the detection of fluorescent molecules at sub-micromolar concentration. Here we report the demonstration of a hollow-core photonic crystal fiber (HC-PCF) probe for remote fluorescence sensing with single molecule sensitivity down to nanomolar concentrations, where both the excitation and fluorescence beams are counter-propagating through the same fiber. A 20 µm polystyrene microsphere is used to efficiently excite and collect the fluorescence from the sample solution thanks to a photonic nanojet effect. Compared to earlier work with silica fibers, the new HC-PCF-microsphere probe achieves a 200x improvement of the signal-to-noise ratio for a single molecule detection event, and a 1000x reduction of the minimum detectable concentration. The device is implemented with fluorescence correlation spectroscopy to distinguish between molecules of similar fluorescence spectra based on the analysis of their translational diffusion properties, and provides similar performance as conventional confocal microscopes.

Free-standing guided-mode resonance band-pass filters: from 1D to 2D structures.

We study experimentally and theoretically band-pass filters based on guided-mode resonances in free-standing metal-dielectric structures with subwavelength gratings. A variety of filters are obtained: polarizing filters with 1D gratings, and unpolarized or selective filters with 2D gratings, which are shown to behave as two crossed-1D structures. In either case, a high transmission (up to ≈ 79 %) is demonstrated, which represents an eight-fold enhancement compared to the geometrical transmission of the grating. We also show that the angular sensitivity strongly depends on the rotation axis of the sample. This behavior is explained with a detailed description of the guided-mode transmission mechanism.

Kagome hollow-core photonic crystal fiber probe for Raman spectroscopy.

We demonstrate the use of a large-pitch Kagome-lattice hollow-core photonic crystal fiber probe for Raman spectroscopy. The large transmission bandwidth of the fiber enables both the excitation and Raman beams to be transmitted through the same fiber. As the excitation beam is mainly transmitted through air inside the hollow core, the silica luminescence background is reduced by over 2 orders of magnitude as compared to standard silica fiber probes, removing the need for fiber background subtraction.

Optical extinction in a single layer of nanorods.

We demonstrate that almost 100% of incident photons can interact with a monolayer of scatterers in a symmetrical environment. Nearly perfect optical extinction through free-standing transparent nanorod arrays has been measured. The sharp spectral opacity window, in the form of a characteristic Fano resonance, arises from the coherent multiple scattering in the array. In addition, we show that nanorods made of absorbing material exhibit a 25-fold absorption enhancement per unit volume compared to unstructured thin film. These results open new perspectives for light management in high-Q, low volume dielectric nanostructures, with potential applications in optical systems, spectroscopy, and optomechanics.

Photonic nanojet focusing for hollow-core photonic crystal fiber probes.

Large-pitch kagome-lattice hollow-core photonic crystal fibers (HC-PCFs) offer appealing optical properties for beam delivery and remote sensing. However, focusing their optical mode on a submicrometer spot can be challenging due to the large mode diameter and low numerical aperture of these fibers. Here, we demonstrate that a 30 µm latex microsphere directly set at the HC-PCF end-face provides an efficient means to focus the fiber mode down to a spot of 540 nm full width at half-maximum thanks to a photonic nanojet effect. The system is used for fluorescence imaging and direct laser writing on a thin absorbing layer. Potential applications include inspection of semiconductor wafers, photolithography, laser surgery, fluorescence sensing, or optical transfection.

λ³/1000 plasmonic nanocavities for biosensing fabricated by soft UV nanoimprint lithography.

Arrays of plasmonic nanocavities with very low volumes, down to λ(3)/1000, have been fabricated by soft UV nanoimprint lithography. Nearly perfect omnidirectional absorption (3-70°) is demonstrated for the fundamental mode of the cavity (λ ≃ 1.15 µm). The second-order mode exhibits a sharper resonance with strong angular dependence and total optical absorption when the critical coupling condition is fulfilled (45-50°, λ ≃ 750 nm). It leads to high refractive index sensitivity (405 nm/RIU) and figure of merit (∼21) and offers new perspectives for efficient biosensing experiments in ultralow volumes.

Guided mode resonance in subwavelength metallodielectric free-standing grating for bandpass filtering.

We present the experimental study of a free-standing metallic guided-mode resonant structure, for bandpass filtering applications in the mid-IR wavelength range. Structure consists of a subwavelength gold grating with narrow slits deposited on a silicon nitride membrane. High optical transmission is measured with up to 78% transmission at resonance. Angularly resolved spectra are presented revealing Fano-type resonance.

Laser-driven radiation: Biomarkers for molecular imaging of high dose-rate effects.

Recently developed short-pulsed laser sources garner high dose-rate beams such as energetic ions and electrons, x rays, and gamma rays. The biological effects of laser-generated ion beams observed in recent studies are different from those triggered by radiation generated using classical accelerators or sources, and this difference can be used to develop new strategies for cancer radiotherapy. High-power lasers can now deliver particles in doses of up to several Gy within nanoseconds. The fast interaction of laser-generated particles with cells alters cell viability via distinct molecular pathways compared to traditional, prolonged radiation exposure. The emerging consensus of recent literature is that the differences are due to the timescales on which reactive molecules are generated and persist, in various forms. Suitable molecular markers have to be adopted to monitor radiation effects, addressing relevant endogenous molecules that are accessible for investigation by noninvasive procedures and enable translation to clinical imaging. High sensitivity has to be attained for imaging molecular biomarkers in cells and in vivo to follow radiation-induced functional changes. Signal-enhanced MRI biomarkers enriched with stable magnetic nuclear isotopes can be used to monitor radiation effects, as demonstrated recently by the use of dynamic nuclear polarization (DNP) for biomolecular observations in vivo. In this context, nanoparticles can also be used as radiation enhancers or biomarker carriers. The radiobiology-relevant features of high dose-rate secondary radiation generated using high-power lasers and the importance of noninvasive biomarkers for real-time monitoring the biological effects of radiation early on during radiation pulse sequences are discussed.

Dose calculations in a cell monolayer for high-throughput irradiation with proton beams generated by PW lasers for space applications.

One of the specific properties of laser-driven radiation is a broadband energy spectrum, which is also a feature of the space radiation fields. This property can be used in materials science studies or radiobiology experiments to simulate the energy spectrum of space radiation exposures in a ground-based laboratory. However, the differences in effects between the higher dose rates of laser generated radiation and the lower dose rates of space radiation have to be investigated in separate, prior studies. A design for a high-throughput irradiation experiment and the associated Monte Carlo dose calculations for a broadband energy proton beam depositing energy in a cell monolayer is presented. Dose control and dose uniformity in the cell monolayer was achieved in the simulations using a variable thickness Ni attenuator. A set of target doses from 0.2 Gy to 4 Gy was obtained and dose uniformity was optimized to less than 4% variability. This work opens the possibility of single or multiple exposures, controllable, high-throughput irradiation experiments on biological samples or materials, using broadband energy particle beams generated by lasers, with relevance for space applications.

Plasmonic antennas and zero-mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy toward physiological concentrations.

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.

Detection of plasmon-enhanced luminescence fields from an optically manipulated pair of partially metal covered dielectric spheres.

Using optical tweezers combined with luminescence measurements we detected the optical field around two optically trapped silica microspheres partially covered by metal. By monitoring the luminescence of rhodamine 6G we were able to observe an increase of the local field intensity owing to the coupling of the local surface plasmons at the surfaces of two spheres.

Spectroscopic mode mapping of resonant plasmon nanoantennas.

We present spatially resolved spectral mode mapping of resonant plasmon gap antennas using two-photon luminescence microspectroscopy. The obtained maps are in good agreement with 3D calculations of the antenna modes. The evolution of the modal field with wavelength, both in the gap and along the two coupled gold nanowires forming the antenna, is directly visualized. At resonance, the luminescence for the gap area is enhanced at least 80 times and a comparison with the antenna extremities shows a dynamical charge redistribution due to the near-field coupling between the two arms.

Cumulative plasmon field enhancement in finite metal particle chains.

The dramatic field enhancement at the extremity of finite chains of strongly coupled gold nanoparticles illuminated under total internal reflection is investigated numerically. We demonstrate that high enhancement factors can be achieved by exploiting the in-plane forward scattering of the particles, with geometries achievable by state-of-the-art lithographic techniques.