RESUMO
We describe the operating principle and performance of a recently developed surface plasmon-enhanced optical sensor that utilizes two-photon excited luminescence of a planar gold film as the reporter signal. The sensor enables direct visualization of nanoscopic binding events near a sensing surface. Light is coupled to the Au/sample interface in an objective-based Kretschmann configuration to excite surface plasmon polariton (SPP) modes at a metal-dielectric interface. The gold luminescence induced by the confined optical field between the particle and the film is detected in the epi-direction by a far-field camera where individual binding events show up as diffraction limited bright spots against a dark background. We study the sensor's emission spectrum and the distance dependence between the target and substrate, which both suggest that the optical signal of the sensor originates from electron-hole pair excitations in the planar Au film. In addition, we show that the well-behaved pointspread function of the sensor enables a straightforward implementation of super-resolution techniques. Finally, we demonstrate the utility of the sensor for detecting DNA binding events, underlining the sensor's usefulness for label-free imaging of nanoscopic particles and biomolecular interactions.
Assuntos
DNA/análise , Ouro/química , Microscopia de Fluorescência/métodos , Algoritmos , DNA/química , DNA/metabolismo , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Rodaminas/química , Razão Sinal-Ruído , Ressonância de Plasmônio de SuperfícieRESUMO
We describe the development and performance of a new type of optical sensor suitable for registering the binding/dissociation of nanoscopic particles near a gold sensing surface. The method shares similarities with surface plasmon resonance microscopy but uses a completely different optical signature for reading out binding events. This new optical read-out mechanism, which we call confined optical field enhanced fluorescence emission (Cofefe), uses pulsed surface plasmon polariton fields at the gold/liquid interface that give rise to confined optical fields upon binding of the target particle to the gold surface. The confined near-fields are sufficient to induce two-photon absorption in the gold sensor surface near the binding site. Subsequent radiative recombination of the electron-hole pairs in the gold produces fluorescence emission, which can be captured by a camera in the far-field. Bound nanoparticles show up as bright confined spots against a dark background on the camera. We show that the Cofefe sensor is capable of detecting gold and silicon nanoparticles, as well as polymer nanospheres and sub-µm lipid droplets in a label-free manner with average illumination powers of less than 10 µW/µm2.
RESUMO
A new method is presented for visualizing the electric field distributions associated with propagating surface-plasmon-polariton (SPP) modes directly in the near-field. The method is based on detecting the photo-induced gradient force exerted by the evanescent field onto a sharp and polarizable tip. Using a photo-induced force microscope (PiFM), images of propagating SPPs are obtained on flat gold surfaces.
RESUMO
We demonstrate rapid vibrational imaging based on sum frequency generation (SFG) microscopy with a collinear excitation geometry. Using the tunable picosecond pulses from a high-repetition-rate optical parametric oscillator, vibrationally selective imaging of collagen fibers is achieved with submicrometer lateral resolution. We furthermore show simultaneous SFG and second harmonic generation imaging to emphasize the compatibility of the microscope with other nonlinear optical modalities.
RESUMO
We demonstrate that four-wave mixing (FWM) signals from individual Si nanoparticles can be generated by the surface fields of traveling surface plasmon polariton modes. We have chosen a counterpropagating excitation scheme in which the nanoparticle is exposed only to surface excitation fields and not to direct laser illumination. We show that background-free, surface-mediated FWM of nanoparticles can be acquired, and that the resulting nonlinear radiation is coherent.
RESUMO
Rigorous calculations are performed to study the effective reduction of the nonlinear excitation volumes when using phase-only masks to condition the pump and Stokes driving fields. Focal volume reduction was achieved using both a multiplicative operation of the excitation fields as well as a subtractive operation. Using a tunable optical bottle beam for the Stokes field, an effective reduction of the width of the excitation volume by a factor of 1.5 can be achieved in the focal plane. Further reduction of the focal volume introduces a rapid growth of sidelobes, which renders such volumes unsuitable for imaging applications. In addition, phase sensitive detection was found to provide information from selective sub-divisions of the engineered coherent anti-Stokes Raman scattering excitation volume. In the case of isolated nanoparticles, an apparent resolution improvement by a factor of 3 is demonstrated, and it is shown that the size of sub-diffraction-limited particles can be accurately determined using phase sensitive detection.
Assuntos
Microscopia/métodos , Análise Espectral Raman/métodos , Técnica de Subtração , Cor , Eletrônica , Modelos Teóricos , Dinâmica não LinearRESUMO
Coherent anti-Stokes Raman scattering (CARS) microscopy is used to determine the distribution and concentration of selected compounds in intact human hair. By generating images based on ratiometric CARS contrast, quantitative concentration maps of both water and externally applied d-glycine are produced in the cortex of human hair fibers. Both water and d-glycine are found to homogeneously distribute throughout the cortical regions of the hair. The ability to selectively detect molecular agents in hair fibers is of direct relevance to understanding the chemical and physical mechanisms that underlie the performance of hair-care products.
Assuntos
Algoritmos , Água Corporal/química , Cabelo/química , Microscopia/métodos , Testes Cutâneos/métodos , Análise Espectral Raman/métodos , HumanosRESUMO
Second-harmonic generation (SHG) imaging is combined with coherent anti-Stokes Raman scattering (CARS) microscopy to follow the process of optical clearing in human skin ex vivo using dimethyl sulfoxide (DMSO) as the optical clearing agent. SHG imaging revealed that DMSO introduces morphological changes to the collagen I matrix. By carefully measuring the dynamic tissue attenuation of the coherent nonlinear signal, using CARS reference signals during the clearing process, it is found that DMSO reduces the overall SHG response from dermal collagen. Evidence is provided for a role of DMSO in compromising the structure of collagen fibers, associated with a reduction of the tissue's scattering properties.
Assuntos
Dimetil Sulfóxido/farmacocinética , Microscopia/métodos , Pele/metabolismo , Análise Espectral Raman , Cadáver , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestrutura , Dimetil Sulfóxido/farmacologia , Humanos , Dinâmica não LinearRESUMO
Coherent anti-Stokes Raman scattering (CARS) interferometry is used to deplete the anti-Stokes radiation emerging from a tightly focused spot. Near-to-complete depletion of the anti-Stokes radiation is obtained when a phase-controlled local oscillator field at the anti-Stokes frequency is out of phase with the induced CARS field in the focal volume. Unlike in traditional interferometry, this depleted energy is not spatially redistributed. A theoretical analysis shows that the energy loss in the anti-Stokes channel is accompanied by an energy gain in the pump and Stokes channels. Interferometric switching of anti-Stokes radiation may offer a route toward developing high-resolution CARS microscopy.
RESUMO
Intrinsic coherent anti-Stokes emission is observed in lithographically patterned gold nanowires. Polarization dependent measurements reveal that the nanostructure's anti-Stokes response is polarized in the direction of the transverse surface plasmon resonance of the wire. We have used specially fabricated gold nanozigzag wires that are modulated in height between 20 and 80 nm to demonstrate tuning of the plasmon polarizability through control of wire height. Stronger anti-Stokes emission is shown to correlate with structures that support higher plasmon polarizability, underlining the primary role of the transverse plasmon resonance in the generation of anti-Stokes radiation from gold nanostructures. Our results also point out that a potential surface-enhanced coherent anti-Stokes Raman scattering (CARS) assay for detecting the vibrational response of surface-tethered molecules needs to include a mechanism for separating the molecular response from the strong intrinsic anti-Stokes emission of the metallic nanosubstrate.
RESUMO
The coherent anti-Stokes Raman scattering (CARS) signal is calculated as a function of focal-field distributions with engineered phase jumps. We show that the focal fields in CARS microscopy can be shaped such that the signal from the bulk is suppressed in the forward detection mode. We present the field distributions that display enhanced sensitivity to vibrationally resonant object interfaces in the lateral dimension. The use of focus-engineered CARS provides a simple means to detect chemical edges against the strong background signals from the bulk.
Assuntos
Algoritmos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia/métodos , Modelos Biológicos , Análise Espectral Raman/métodos , Tomografia de Coerência Óptica/métodos , Simulação por Computador , Aumento da Imagem/instrumentação , Interpretação de Imagem Assistida por Computador/instrumentação , Microscopia/instrumentação , Análise Numérica Assistida por Computador , Análise Espectral Raman/instrumentação , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
We report a simple and robust computer-based active interferometer stabilization scheme which does not require modulation of the interfering beams and relies on an error signal which is linearly related to the optical path difference. In this setup, a non-collinearly propagating reference laser beam stabilizes the interference output of the laser light propagating collinearly through the interferometer. This stabilization scheme enables adjustable phase control with 20 ms switching times in the range from 0.02pi radians to 6pi radians at 632.8 nm.
RESUMO
We generate tunable picosecond anti-Stokes pulses by four-wave mixing of two picosecond pump and Stokes pulse trains in a photonic-crystal fiber. The visible, spectrally narrow anti-Stokes pulses with shifts over 150 nm are generated without generating other spectral features. As a demonstration, we employ the generated anti-Stokes pulses as reference pulses in an interferometric coherent anti-Stokes Raman scattering imaging experiment showing that interpulse coherence among the pump, Stokes and anti-Stokes beams is retained.