Showing papers by "Hervé Rigneault published in 2010"

Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection

Abstract: Recent advances in nanophotonics open the way for promising applications towards efficient single molecule fluorescence analysis. In this review, we discuss how photonic methods bring innovative solutions for two essential questions: how to detect a single molecule in a highly concentrated solution, and how to enhance the faint optical signal emitted per molecule? The focus is set primarily on the widely used technique of fluorescence correlation spectroscopy (FCS), yet the discussion can be extended to other single molecule detection methods.

Surface Enhanced Raman Scattering on a Single Nanometric Aperture

Abstract: Arrays of nanoapertures have been demonstrated to realize efficient, robust, and reproducible substrates for surface-enhanced Raman scattering SERS spectroscopy. However, little attention has been devoted to single nanoapertures, although a thorough understanding of the SERS phenomenon in a single aperture is essential for the rationale optimization of nanoaperture arrays SERS. In this study, single nanoapertures milled in optically thick gold films are quantitatively evaluated for the first time to determine the SERS enhancement factors using para-mercaptoaniline as nonresonant analyte. We determine a peak enhancement factor of 2 × 105 for a single 100 nm diameter aperture. Although this is a moderate enhancement factor, we believe that nanoapertures deserve special attention to highlight the physical and chemical phenomena leading to SERS enhancement and better understand the design of nanoaperture arrays for SERS substrates. The experimental data are supported by numerical simulations and argue for a c...

Enhanced second-harmonic generation from individual metallic nanoapertures

Abstract: We demonstrate the ability of single-subwavelength-size nanoapertures fabricated in a gold metal thin film to enhance second-harmonic generation (SHG) as compared to a bare metal film Nonlinear microscopy imaging with polarization resolution is used to quantify the SHG enhancement in circular and triangular nanoaperture shapes The dependence of the measured SHG enhancement on circular aperture diameters is seen to originate from both phase retardation effects and field enhancements at the nanoaperture edge Triangular nanoapertures exhibit superior SHG enhancement compared with circular ones, as expected from their noncentrosymmetric shape

Polarization and phase pulse shaping applied to structural contrast in nonlinear microscopy imaging

Abstract: The spectral polarization shaping of ultrashort pulses is shown to allow retrieval of two-dimensional individual tensorial components of the second-harmonic-generation response of molecular samples in nonlinear microscopy imaging. This configuration, which cannot be performed by traditional polarization-controlled excitation, provides a structural contrast that can be directly related to information on the local symmetry and order of the sample, with submicrometric spatial resolution. Phase shaping, in addition to polarization spectral manipulation, is proposed as a possible scheme for imaging individual tensorial components without the need for a spectral information extraction.

Colloidal quantum dots as probes of excitation field enhancement in photonic antennas.

Abstract: Optical antennas are essential devices to interface light to nanoscale volumes and locally enhance the electromagnetic intensity. Various experimental methods can be used to quantify the antenna amplification on the emission process, yet characterizing the antenna amplification at the excitation frequency solely is a challenging task. Such experimental characterization is highly needed to fully understand and optimize the antenna response. Here, we describe a novel experimental tool to directly measure the antenna amplification on the excitation field independently of the emission process. We monitor the transient emission dynamics of colloidal quantum dots and show that the ratio of doubly to singly excited state photoluminescence decay amplitudes is an accurate tool to quantify the local excitation intensity amplification. This effect is demonstrated on optical antennas made of polystyrene microspheres and gold nanoapertures, and supported by numerical computations. The increased doubly excited state fo...

High order symmetry structural properties of vibrational resonances using multiple-field polarization coherent anti-Stokes Raman spectroscopy microscopy.

Abstract: Polarization-resolved coherent anti-Stokes Raman scattering (CARS) is usually applied to measure the depolarization ratio in solutions or evidence orientation effects in anisotropic media. We present an extensive approach based on multiple-field polarization-resolved CARS, in order to unravel the complexity of vibrational resonances up to the fourth-order symmetry, at the microscopic scale in nonisotropic media. The CARS polarized signals measured under a continuous variation of the incident pump and/or Stokes excitation beams are analyzed using a full tensorial picture both in the nonresonant and resonant regimes. This method evidences the strong influence of vibrational symmetries on polarized CARS, and more specifically the occurrence of Kleinman symmetry deviations at the vicinity of the Raman lines frequencies. This technique, illustrated on a cubic symmetry crystal, is general and can be applied to other medium symmetries.

Revisiting the Young's Double Slit Experiment for Background-Free Nonlinear Raman Spectroscopy and Microscopy

Abstract: In the Young's double slit experiment, the spatial shift of the interference pattern projected onto a screen is directly related to the phase difference between the fields diffracted by the two slits. We apply this property to fields emitted by nonlinear processes and thus demonstrate background-free coherent anti-Stokes Raman scattering microscopy near an axial interface between a resonant and a nonresonant medium. This method is relevant to remove the nonresonant background in other coherent resonant processes.

Two-photon fluorescence correlation spectroscopy with high count rates and low background using dielectric microspheres

Abstract: Two-photon excitation fluorescence is a powerful technique commonly used for biological imaging. However, the low absorption cross section of this non-linear process is a critical issue for performing biomolecular spectroscopy at the single molecule level. Enhancing the two-photon fluorescence signal would greatly improve the effectiveness of this technique, yet current methods struggle with medium enhancement factors and/or high background noise. Here, we show that the two-photon fluorescence signal from single Alexa Fluor 488 molecules can be enhanced up to 10 times by using a 3 µm diameter latex sphere while adding almost no photoluminescence background. We report a full characterization of the two-photon fluorescence enhancement by a single microsphere using fluorescence correlation spectroscopy. This opens new routes to enhance non-linear optical signals and extend biophotonic applications.

Enhanced fluorescence from metal nanoapertures: physical characterizations and biophotonic applications

Abstract: Nanoapertures milled in opaque metallic films offer a simple and robust photonic tool to significantly enhance the fluorescence of single molecules. We provide a detailed physical characterization of this phenomenon for apertures milled in gold and aluminum, and discuss its application to biophotonics. For the first time, the most general figures are provided to predict the awaited enhancement factors for almost every kind of fluorescent molecule. This knowledge is essential to discuss the ability to detect low-quantum yield species. We also report the first demonstration of single metal nanoapertures to perform DNA hybridization sensing, and measure similar enhancement factors as for experiments on diffusing molecules.

High-efficiency single molecule fluorescence detection and correlation spectroscopy with dielectric microspheres

Abstract: Microspheres made of high refractive index melamine resin are shown to enhance the fluorescence from single molecules in solution by seven-fold, and simultaneously reduce the observation volume by thirteen-fold, as compared to state-of-the-art confocal microscopy. This fluorescence enhancement is demonstrated to dramatically increase the signal-to-noise ratio in fluorescence correlation spectroscopy and reduce the experiment integration time by fifty-fold. We also provide the first description of dual-color fluorescence cross-correlation spectroscopy (FCCS) enhanced by a dielectric microsphere, and report comparable enhancement factors as for the single color case.

Polarization and Phase Pulse Shaping applied to nonlinear tensorial structure read-out

Abstract: The spectral polarization shaping of ultra-short pulses is shown to allow retrieval of 2D individual tensorial components of the Second Harmonic Generation response of crystalline samples.

Fluorescence correlation spectroscopy system for analyzing particles in a medium

Abstract: The present invention relates to a fluorescence correlation spectroscopy system (1) for analyzing particles in a medium (2), including a means (3) for detecting the light (7) emitted by the particles in the medium (2), said means (3) being coupled to a waveguide (4), for which purpose the end piece of the guide (4) comprises a means (4b; 5) for confining the light (7) injected into the guide (4).

Pixel-type biological analysis device, biosensor, and fabrication processes thereof

Abstract: The disclosure relates to a pixel-type biological analysis device comprising a photosensitive layer, a capture mixture for the capture of targeted proteins, the capture mixture being placed on an external surface of the photosensitive layer and comprising a protein probe grafted to a hydrogel, collection means for collecting photoelectrons in the photosensitive layer, and reading and treatment means of an electrical quantity supplied by the collection means, for the supply of a characteristic value of a luminous intensity detected by the photosensitive layer.

Fabrication process of a biosensor on a semiconductor substrate

Abstract: The disclosure relates to a fabrication process of a biosensor on a semiconductor wafer, comprising steps of: making a central photosensitive zone comprising at least one pixel-type biological analysis device comprising a photosensitive layer, and a first peripheral zone surrounding the central photosensitive zone, comprising electronic circuits. The first peripheral zone is covered by a hydrophilic coating, and the central photosensitive zone is covered with a hydrophobic coating. A barrier of a bio-compatible resin is formed on the second peripheral zone.

Quantifying molecular order in biological samples using polarimetric muti-modal nonlinear microscopy

Abstract: In addition to their interesting capabilities for bio-imaging, nonlinear optical contrasts involve rich excitation/emission processes based on multiple fields and therefore multiple polarization directions coupling. This opens the way to analysis of high order symmetry features in bio-molecular assemblies down to the nanometric scale, that are not accessible by linear optics tools [1]. We have recently developed a general polarimetric nonlinear microscopy technique able to circumvent the intrinsic limitations of fluorescence anisotropy, which is today the most widely used technique to measure molecular order information in biological samples. This polarimetric method, based on a variation of the incident fields polarization states combined with a polarized read-out of the emitted signal, allows retrieving information on local molecular angular distribution with no necessary a priori knowledge on its averaged orientation nor its shape. We will demonstrate on several examples how this polarization-dependent nonlinear microscopy technique is able to measure quantitatively molecular order information in biological samples. Different contrasts will be described with emphasis on their specificities, either for incoherent processes such as multiphoton fluorescence or for coherent harmonic generations. These specificities are illustrated in crystalline samples, cells and tissues, where the contrasts are provided either from membrane markers (two-photon fluorescence) [2], from intrinsic mechanisms such as second harmonic generation (SHG) [3] or vibrational signatures using Coherent Anti Stokes Raman Scattering (CARS) [4]. Instrumental issues will be discussed, in particular on the diagnostics of polarization distortions due to complex samples such as tissues [3], or inherent to polarization-resolved microscopy [5].

Broadband multiplex CARS microspectroscopy in the picosecond regime

Abstract: We investigate a compact, stable and broadband multiplex Coherent anti-Stokes Raman Scattering (CARS) source for micro-spectroscopy. By pumping an adapted photonic crystal fiber, we generate the broadband Stokes pulses required for multiplex CARS measurements. The CARS signal stability is provided by an active fiber coupling, avoiding therefore the thermal or mechanical drifts. With only a few nanojoule for pump and Stokes pulses energies, we demonstrate on test liquids the capability of the source to generate multiplex CARS spectra in the 600-2000 cm-1 spectral range.

Broadband multiplex CARS micro-spectroscopy in the picosecond regime

Abstract: We investigate a compact, stable and broadband multiplex Coherent anti-Stokes Raman Scattering (CARS) source for micro-spectroscopy. By pumping an adapted photonic crystal fiber, we generate the broadband Stokes pulses required for multiplex CARS measurements. The CARS signal stability is provided by an active fiber coupling, avoiding therefore the thermal or mechanical drifts. With only a few nanojoule for pump and Stokes pulses energies, we demonstrate on test liquids the capability of the source to generate multiplex CARS spectra in the 600-2000 cm-1 spectral range.

A method for detecting an optical signal and nonlinear resonant device for implementation of said method

Abstract: The invention relates to a method and a device for detecting a nonlinear optical resonant signal induced in a sample (805) comprising a resonating medium (61) and a nonresonant medium forming an interface, the device comprising: - an emission source (801) at least a first excitation light beam of the resonant medium, said pump beam at a first ωp given pulse, said pump beam is incident on the sample along an optical axis, and intercepting the sample to a given position of a transverse interface between the resonant and nonresonant medium, - a first optical module (803) for detecting the non-linear optical signal resulting from the interaction of said beams or with the sample, - reflecting means ( 813) or of said excitation beams, the one or more excitation beams thus reflected intercepting said transverse interface at substantially the same position as the beam or beams of Incident excitation, - a second optical module (806) for detecting the non-linear optical signal resulting from the interaction of said excitation or reflected beams with the sample, - a processing module (830) optical signals detected by said first and second detecting modules, comprising calculating a difference of the detected signals, the difference being characteristic of a vibrational resonance of the resonant or electronic medium.

Polarization-resolved coherent anti-Stokes Raman scattering microscopy

Abstract: We have implemented a polarization-resolved coherent anti-Stokes Raman scattering (CARS) microscopy, based on the continuous variation of the incident linear polarization at the pump and Stokes wavelengths, together with a polarized analysis of the anti-Stokes signal. In isotropic media, such as solutions, this technique can be a powerful way to probe microscopic-scale information, such as the vibrational symmetry properties of the molecular bonds. In ordered media, additional macroscopic-scale structural information can be obtained, such as the orientation of the unit-cell of a crystal in 3D.

Système de spectroscopie de fluorescence par corrélation temporelle pour l'analyse de particules dans un milieu

Abstract: La presente invention concerne un systeme de spectroscopie (1) de fluorescence par correlations temporelles pour l'analyse de particules dans un milieu (2), comprenant un moyen de detection (3) de la lumiere (7) emise par les particules dans le milieu (2), le moyen (3) etant couple a un guide d'onde (4), pour lequel le guide (4) comporte en son embout (4a) un moyen de confinement (4b;5) de la lumiere (7) injectee dans le guide (4).