Hervé Rigneault

Single molecule fluorescence in rectangular nano-apertures

Fluorescence Correlation Spectroscopy is used to investigate fluorescent molecules in solution diffusing in subwavelength rectangular apertures milled in Aluminium films. This rectangular shape allows to switch between a propagating and an evanescent excitation field within the aperture, leading to a significant tunability of the observation volume. Due to the vicinity of the metal surface, the fluorophores molecular lifetime inside the aperture appears to be dramatically reduced whatever the excitation field is set to. However, for a properly tailored evanescent excitation field within the nanoaperture, the detected fluorescence rate per molecule is significantly enhanced as compared to open solution. This suggests that the observed molecular fluorescence enhancement is mainly due to the excitation near field within the subwavelength aperture.

Single-Fluorophore Diffusion in a Lipid Membrane over a Subwavelength Aperture

We use submicrometer apertures milled in an aluminium film to study the diffusion dynamics of β-Bodipy-FL-C5-HPC (Bodipy-PC) fluorophores in a lipid dioleoylphosphatidylcholine (DOPC) multilayer. The observation volume is limited by the aperture diameter, well below the optical wavelength. This spatial resolution improvement comes together with an enhancement of the detected fluorescence per molecule as compared to an open sample, with a significant increase up to 3.5 times.

Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations

Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence cross-correlation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 muM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoaper-tures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.

Coherent anti-Stokes Raman scattering (CARS) microscopy imaging at interfaces: evidence of interference effects

We show in this paper that the contrast of the interface between resonant and nonresonant media imaged in Coherent anti-Stokes Raman scattering (CARS) microscopy strongly depends on the pump and Stokes fields spectral detuning. More specifically, when this detuning drives the vibrational resonance with the maximum phase difference, a spatial dip appears at the interface in the CARS image. This effect is studied both theoretically and experimentally and is an evidence of the coherent and resonant nature of the CARS contrast mechanism.

Light-Assisted Solvothermal Chemistry Using Plasmonic Nanoparticles

Solvothermal synthesis, denoting chemical reactions occurring in metastable liquids above their boiling point, normally requires the use of a sealed autoclave under pressure to prevent the solvent from boiling. This work introduces an experimental approach that enables solvothermal synthesis at ambient pressure in an open reaction medium. The approach is based on the use of gold nanoparticles deposited on a glass substrate and acting as photothermal sources. To illustrate the approach, the selected hydrothermal reaction involves the formation of indium hydroxide microcrystals favored at 200 °C in liquid water. In addition to demonstrating the principle, the benefits and the specific characteristics of such an approach are investigated, in particular, the much faster reaction rate, the achievable spatial and time scales, the effect of microscale temperature gradients, the effect of the size of the heated area, and the effect of thermal-induced microscale fluid convection. This technique is general and could be used to spatially control the deposition of virtually any material for which a solvothermal synthesis exists.

Background-free coherent anti-Stokes Raman spectroscopy near transverse interfaces: a vectorial study

A full-vectorial theoretical investigation of the recently proposed background-free coherent anti-Stokes Raman scattering (CARS) spectroscopy near transverse interfaces [Phys. Rev. A, 77, 061802(R) (2008)] is presented. In this scheme, the field symmetries of the focused excitation beams are applied to recover the pure Raman spectrum of a medium forming a transverse interface with a nonresonant medium. We show that this method is robust to spatial shift of the excitation volume relative to the interface and does not depend on the linear polarizations states of the pump and Stokes beams. Finally, we extend the applicability of this scheme to a succession of planar interfaces between resonant and nonresonant media, which is potentially interesting for fast chemical analysis in microfluidic devices. Interestingly, this method can be extended to nondegenerated resonant four-wave-mixing processes to remove the nonresonant background.

Coherent anti-Stokes Raman scattering in a Fabry-Perot cavity: A theoretical study

We present a study of coherent anti-Stokes Raman scattering (CARS) under focused beam excitation in a planar Fabry-Perot cavity using an image-dipole formalism. We give a comprehensive description of forward- and backward-CARS signal generation by introducing the expressions of the nonlinear induced polarization in the cavity as a function of their counterpart in free space. We show that the cavity gives rise to a backward-CARS signal and allows working with low numerical aperture collection objectives. We finally discuss the influence of the scatterer position in the cavity on the detected signal.

In-plane plasmonic antenna arrays resolve nanoscopic phase separation in model lipid membranes (Conference Presentation)

Resolving the various interactions of lipids and proteins in the plasma membrane of living cells with high spatiotemporal resolution is of upmost interest [1]. Here we introduce an innovative design of plasmonic nanogap antennas to monitor single-molecule events on model biological membranes at physiological relevant concentrations by means of fluorescence correlation spectroscopy. Our design involves the fabrication of in-plane plasmonic nanogap antennas arrays embedded in nanometric-size boxes to provide full surface accessibility of the hotspot-confined region. Using these antennas we recently reported fluorescence enhancement factors of 104-105 times on individual molecules diffusing in solution, together with nanoscale detection volumes in the zeptoliter range [2]. In principle, the planarity of these antennas should enable similar studies on biological membranes without unwanted membrane curvature effects. To show their applicability, we recorded the diffusion of individual molecules inserted in multi-component lipid bilayers as a simple mimetic system that recapitulates some of the most important features of cell membranes. We prepared membranes of different compositions: saturated phospholipids, sphingolipids and cholesterol and used antennas of different gap sizes (10-45 nm). The diffusion of individual molecules on membranes consisting of phospholipids and/or in a mixture with sphingolipids resulted Brownian, confirming homogenous lipid distribution. Interestingly, the strong confinement of antennas revealed the formation of transient (<1ms lifetime) nanoscopic domains of ~11 nm in size upon cholesterol addition. These results indicate that in-plane antennas represent a highly promising non-invasive tool to investigate the nanoscale dynamic organization of biological membranes and its impact in biological function. References: [1] D. Lingwood, K. Simons, Science 327, 46 (2010). [2] V. Flauraud et al, submitted.