Spectroscopy of individual Brownian nanoparticles in real-time using holographic localization.

Individual nanoparticle spectroscopic characterization is fundamental, but challenging in liquids. While confocal selectivity is necessary to isolate a particle in a crowd, Brownian motion constantly offsets the particle from the light collection volume. Here, we present a system able to acquire holograms and reconstruct them to precisely determine the 3D position of a particle in real time. These coordinates drive an adaptive system comprising two galvanometric mirrors (x,y, transverse directions) and a tunable lens (z, longitudinal) which redirect light scattered from the corresponding region of space towards the confocal entrance of a spectrometer, thus allowing long spectral investigations on individual, freely-moving particles. A study of the movements and spectra of individual 100 nm Au nanoparticles undergoing two types of aggregations illustrates the possibilities of the method.

3D nanoparticle superlocalization with a thin diffuser.

We report on the use of a thin diffuser placed in the close vicinity of a camera sensor as a simple and effective way to superlocalize plasmonic nanoparticles in 3D. This method is based on holographic reconstruction via quantitative phase and intensity measurements of a light field after its interaction with nanoparticles. We experimentally demonstrate that this thin diffuser can be used as a simple add-on to a standard bright-field microscope to allow the localization of 100 nm gold nanoparticles at video rate with nanometer precision (1.3 nm laterally and 6.3 nm longitudinally). We exemplify the approach by revealing the dynamic Brownian trajectory of a gold nanoparticle trapped in various pockets within an agarose gel. The proposed method provides a simple but highly performant way to track nanoparticles in 3D.

Background-suppressed SRS fingerprint imaging with a fully integrated system using a single optical parametric oscillator.

Stimulated Raman Scattering (SRS) imaging can be hampered by non-resonant parasitic signals that lead to imaging artifacts and eventually overwhelm the Raman signal of interest. Stimulated Raman gain opposite loss detection (SRGOLD) is a three-beam excitation scheme capable of suppressing this nonlinear background while enhancing the resonant Raman signal. We present here a compact electro-optical system for SRGOLD excitation which conveniently exploits the idler beam generated by an optical parametric oscillator (OPO). We demonstrate its successful application for background suppressed SRS imaging in the fingerprint region. This system constitutes a simple and valuable add-on for standard coherent Raman laser sources since it enables flexible excitation and background suppression in SRS imaging.

Monitoring Cobalt-Oxide Single Particle Electrochemistry with Subdiffraction Accuracy.

By partially overcoming the diffraction limit, superlocalization techniques have extended the applicability of optical techniques down to the nanometer size-range. Herein, cobalt oxide-based nanoparticles are electrochemically grown onto carbon nanoelectrodes and their individual catalytic properties are evaluated through a combined electrochemical-optical approach. Using dark-field white light illumination, edges superlocalization techniques are applied to quantify changes in particle size during electrochemical activation with down to 20 nm precision. It allows the monitoring of (i) the anodic electrodeposition of cobalt hydroxide material and (ii) the large and reversible volume expansion experienced by the cobalt hydroxide particle during its oxidation. Meanwhile, the particle light scattering provides chemical information such as the Co redox state transformation, which complements both the particle size and the recorded electrochemical current and provides in operando mechanistic information on particle electrocatalytic properties.

Wrapping-free numerical refocusing of scalar electromagnetic fields.

Numerical refocusing in any plane is one powerful feature granted by measuring both the amplitude and the phase of a coherent light beam. Here, we introduce a method based on the first Rytov approximation of scalar electromagnetic fields that (i) allows numerical propagation without requiring phase unwrapping after propagation and (ii) limits the effect of artificial phase singularities that appear upon numerical defocusing when the measurement noise is mixing with the signal. We demonstrate the feasibility of this method with both scalar electromagnetic field simulations and real acquisitions of microscopic biological samples imaged at high numerical aperture.

Electrochemistry of Single Nanodomains Revealed by Three-Dimensional Holographic Microscopy.

Interest in nanoparticles has vigorously increased over the last 20 years as more and more studies show how their use can potentially revolutionize science and technology. Their applications span many different academically and industrially relevant fields such as catalysis, materials science, health, etc. Until the past decade, however, nanoparticle studies mostly relied on ensemble studies, thus leaving aside their chemical heterogeneity at the single particle level. Over the past few years, powerful new tools appeared to probe nanoparticles individually and in situ. This Account describes how we drew inspiration from the emerging fields of nanoelectrochemistry and plasmonics-based high resolution holographic microscopy to develop a coupled approach capable of analyzing in operando (electro)chemical reaction over one single nanoparticle. A brief overview of selected optical strategies to image NPs in situ with emphasis on scattering based methods is presented. In an electrochemical context, it is necessary to track particle behavior both in solution and near a polarized electrode, which is why 3D optical observation is particularly appealing. These approaches are discussed together with strategies to track NPs beyond the diffraction limit, allowing a much finer description of their trajectories. Then, the holographic setup is used to study electrochemically triggered Ag NP oxidation reaction in the presence of different electrolytes. Holography is shown to be a powerful technique to track and analyze the trajectory of individual NPs in situ, which further sheds light on in operando behaviors such as electrogenerated NP transport, aggregation, or adsorption. We then show that spectroscopy and scattering-based optical methods are reliable and sensitive to the point of being used to investigate and quantify NP (electro)chemical reactions in model cases. However, since real chemical reactions usually take place in an inherently complex environment, approaches based exclusively on optical imaging only reach their limitations. The strategy is then taken one step further by merging together electrochemical nanoimpact experiments with 3D optical monitoring. Previous strategies are validated by showing that in simple cases, these two independent ways of probing NP size and reactivity yield the same results. For more complicated reactions (e.g., multistep reactions), one must go beyond either technique by showing that the two approaches are perfectly complementary and that the two signals contain information of different natures, thus providing a much better characterization of the reaction. This point is illustrated by studying Ag NP oxidation (single or agglomerates) in the presence of a precipitating agent, where the actual oxidation is uncoupled from the dissolution of the particle, thus proving the point of our symbiotic approach.

Wavefront sensing with a thin diffuser.

We propose and implement a broadband, compact, and low-cost wavefront sensing scheme by simply placing a thin diffuser in the close vicinity of a camera. The local wavefront gradient is determined from the local translation of the speckle pattern. The translation vector map is computed thanks to a fast diffeomorphic image registration algorithm and integrated to reconstruct the wavefront profile. The simple translation of speckle grains under local wavefront tip/tilt is ensured by the so-called "memory effect" of the diffuser. Quantitative wavefront measurements are experimentally demonstrated, both for the few first Zernike polynomials and for phase-imaging applications requiring high resolution. We finally provided a theoretical description of the resolution limit that is supported experimentally.

Programmable single-pixel-based broadband stimulated Raman scattering.

We report a simple add-on for broadband stimulated Raman scattering (SRS) microscopes to enable fast and programmable spectroscopy acquisition. It comprises a conventional dispersive spectrometer layout incorporating a fast digital micromirror device (DMD). The approach is validated by acquiring SRS spectra of standard chemicals. We demonstrate a DMD's advantage in broadband SRS by showing higher signal-to-noise ratio using a multiplexed Hadamard spectral basis and compressive sensing detection. Our results apply to a variety of frequency-domain pump-probe spectroscopy.

Opto-electrochemical In Situ Monitoring of the Cathodic Formation of Single Cobalt Nanoparticles.

Single-particle electrochemistry at a nanoelectrode is explored by dark-field optical microscopy. The analysis of the scattered light allows in situ dynamic monitoring of the electrodeposition of single cobalt nanoparticles down to a radius of 65 nm. Larger sub-micrometer particles are directly sized optically by super-localization of the edges and the scattered light contains complementary information concerning the particle redox chemistry. This opto-electrochemical approach is used to derive mechanistic insights about electrocatalysis that are not accessible from single-particle electrochemistry.

Electrochemical transformation of individual nanoparticles revealed by coupling microscopy and spectroscopy.

Although extremely sensitive, electrical measurements are essentially unable to discriminate complex chemical events involving individual nanoparticles. The coupling of electrochemistry to dark field imaging and spectroscopy allows the triggering of the electrodissolution of an ensemble of Ag nanoparticles (by electrochemistry) and the inference of both oxidation and dissolution processes (by spectroscopy) at the level of a single nanoparticle. Besides the inspection of the dissolution process from optical scattering intensity, adding optical spectroscopy reveals chemical changes through drastic spectral changes. The behaviours of single NPs and NP agglomerates are differentiated: in the presence of thiocyanate ions, the transformation of Ag single nanoparticles to AgSCN is investigated in the context of plasmonic coupling with the electrode; tentative interpretations for optically unresolved groups of nanoparticles are proposed.

Background-free stimulated Raman spectroscopy and microscopy.

We propose a three-color, double-modulation scheme for the background-free detection of stimulated Raman scattering (SRS). We call the scheme stimulated Raman gain and opposite loss detection (SRGOLD). It exploits the symmetric nature of potential parasitic signals (cross phase modulation, two-photon absorption, and thermal effects) to the end of suppressing them. Conversely, the antisymmetric nature of SRS provides for a twofold increase in the magnitude of the SRS signal. We experimentally demonstrate SRGOLD spectroscopy and microscopy on test samples as well as on mice skin samples.

Single-shot digital optical fluorescence phase conjugation through forward multiple-scattering samples.

Aberrations and multiple scattering in biological tissues critically distort light beams into highly complex speckle patterns. In this regard, digital optical phase conjugation (DOPC) is a promising technique enabling in-depth focusing. However, DOPC becomes challenging when using fluorescent guide stars for four main reasons: the low photon budget available, the large spectral bandwidth of the fluorescent signal, the Stokes shift between the emission and the excitation wavelength, and the absence of reference beam preventing holographic measurement. Here, we demonstrate the possibility to focus a laser beam through multiple-scattering samples by measuring speckle fields in a single acquisition step with a reference-free, high-resolution wavefront sensor. By taking advantage of the large spectral bandwidth of forward multiply scattering samples, digital fluorescence phase conjugation is achieved to focus a laser beam at the excitation wavelength while measuring the broadband speckle field arising from a micrometer-sized fluorescent bead.

Wide-field vibrational phase imaging in an extremely folded box-CARS scattering geometry.

We present a method that allows one to measure the real and imaginary parts of the third-order susceptibility in a wide-field coherent anti-Stokes Raman scattering setup using a quadriwave lateral shearing interferometer. This permits the retrieval of the undistorted Raman spectrum and the removal of a nonresonant signal from the surrounding solvent, which otherwise may overwhelm weak resonances.

Wide-field vibrational phase imaging.

We propose and implement a wide-field microscopy method to retrieve the real and imaginary part of a field emitted by coherent and resonant molecular scatterers. The technique is based on wave-front sensing and does not require the use of any reference beam. We exemplify its ability in wide-field coherent anti-Stokes Raman scattering imaging and retrieve the complex anti-Stokes field while spectrally scanning a molecular vibrational resonance. This approach gives access to the background-free Raman spectrum of the targeted molecular bond.

Local Surface Chemistry Dynamically Monitored by Quantitative Phase Microscopy.

Surface modification by photo grafting constitutes an interesting strategy to prepare functional surfaces. Precision applications, however, demand quantitative methods able to monitor and control the amount and distribution of surface modifications, which is hard to achieve, particularly in operando conditions. In this paper, a label-free, cost-effective, all-optical method based on wavefront sensing which is able to quantitatively track the evolution of grafted layers in real-time, is presented. By positioning a simple thin diffuser in the close vicinity of a camera, the thickness of grafted patterns is directly evaluated with sub-nanometric sensitivity and diffraction-limited lateral resolution. By performing an in-depth kinetic analysis of the local modification of an inert substrate (glass cover slips) through photografting of arydiazonium salts, different growth regimes are characterized and several parameters are estimated, such as the grafting efficiency, density and the apparent refractive index distribution of the resulting grafted layers. Both focused and widefield-grafting can be quantitatively monitored in real time, providing valuable guidelines to maximize functionalization efficiency. The association of a well-characterized versatile photografting reaction with the proposed flexible and sensitive monitoring strategy enables functional surfaces to be prepared, and puts surface micro- to submicro-structuration within the reach of most laboratories.

3D Spectroscopic Tracking of Individual Brownian Nanoparticles during Galvanic Exchange.

Monitoring chemical reactions in solutions at the scale of individual entities is challenging: single-particle detection requires small confocal volumes, which are hardly compatible with Brownian motion, particularly when long integration times are necessary. Here, we propose a real-time (10 Hz) holography-based nm-precision 3D tracking of single moving nanoparticles. Using this localization, the confocal collection volume is dynamically adjusted to follow the moving nanoparticle and allow continuous spectroscopic monitoring. This concept is applied to study galvanic exchange in freely moving colloidal silver nanoparticles with gold ions generated in situ. While the Brownian trajectory reveals particle size, spectral shifts dynamically reveal composition changes and transformation kinetics at the single-object level, pointing at different transformation kinetics for free and tethered particles.

Double-clad hollow core photonic crystal fiber for coherent Raman endoscope.

Performing label free coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) in endoscope imaging is a challenge, with huge potential clinical benefit. To date, this goal has remained inaccessible because of the inherent coherent Raman noise that is generated in the fiber itself. By developing double-clad hollow core photonic crystal fiber, we demonstrate coherent anti-Stokes Raman scattering and stimulated Raman scattering in an 'endoscope-like' scheme. Both the excitation beams and the collected CARS and SRS signals travel through the same fiber. No CARS and SRS signals are generated within the hollow core fiber even for temporally overlapping pump and Stokes beams, leading to excellent image quality. The CARS and SRS signals generated in the sample are coupled back into a high numerical aperture multimode cladding surrounding the central photonic crystal cladding. We demonstrate this scheme by imaging molecular vibrational bonds of organic crystal deposited on a glass surface.

Stimulated Raman scattering microscopy by spectral focusing and fiber-generated soliton as Stokes pulse.

We demonstrate stimulated Raman microscopy with broadband pump and Stokes pulses, using spectral focusing to attain spectral resolution and to rapidly acquire spectra within a spectral window determined by the bandwidth of the pulses. As the Stokes pulse, we use the redshifted soliton generated in a photonic crystal fiber, which allows for simple shifting of the accessible spectral window.

Phototoxic damage to cone photoreceptors can be independent of the visual pigment: the porphyrin hypothesis.

Lighting is rapidly changing with the introduction of light-emitting diodes (LEDs) in our homes, workplaces, and cities. This evolution of our optical landscape raises major concerns regarding phototoxicity to the retina since light exposure is an identified risk factor for the development of age-related macular degeneration (AMD). In this disease, cone photoreceptors degenerate while the retinal pigment epithelium (RPE) is accumulating lipofuscin containing phototoxic compounds such as A2E. Therefore, it remains unclear if the light-elicited degenerative process is initiated in cones or in the RPE. Using purified cone photoreceptors from pig retina, we here investigated the effect of light on cone survival from 390 to 510 nm in 10 nm steps, plus the 630 nm band. If at a given intensity (0.2 mW/cm²), the most toxic wavelengths are comprised in the visible-to-near-UV range, they shift to blue-violet light (425-445 nm) when exposing cells to a solar source filtered by the eye optics. In contrast to previous rodent studies, this cone photoreceptor phototoxicity is not related to light absorption by the visual pigment. Despite bright flavin autofluorescence of cone inner segment, excitation-emission matrix of this inner segment suggested that cone phototoxicity was instead caused by porphyrin. Toxic light intensities were lower than those previously defined for A2E-loaded RPE cells indicating cones are the first cells at risk for a direct light insult. These results are essential to normative regulations of new lighting but also for the prevention of human retinal pathologies since toxic solar light intensities are encountered even at high latitudes.

Quantification of gold nanoparticle accumulation in tissue by two-photon luminescence microscopy.

Nanomedicine has emerged as a promising strategy to address some of the limitations of traditional biomedical sensing, imaging and therapy modalities. Its applicability and efficacy are, in part, hindered by the difficulty in both controllably delivering nanoparticles to specific regions and accurately monitoring them in tissue. Gold nanoparticles are among the most extensively used inorganic nanoparticles which benefit from high biocompatibility, flexible functionalization, strong and tunable resonant absorption, and production scalability. Moreover, their capability to enhance optical fields at their plasmon resonance enables local boosting of non-linear optical processes, which are otherwise very inefficient. In particular, two-photon induced luminescence (TPL) in gold offers high signal specificity for monitoring gold nanoparticles in a biological environment. In this article, we demonstrate that TPL microscopy provides a robust sub-micron-resolution technique able to quantify accumulated gold nanorods (GNRs) both in cells and in tissues. First, the temporal accumulation of GNRs with two different surface chemistries was measured in 786-O cells during the first 24 hours of incubation, and at different nanoparticle concentrations. Subsequently, GNR accumulation in mice, 6 h and 24 hours after tail vein injection, was quantified by TPL microscopy in biopsied tissue from kidney, spleen, liver and clear cell renal cell carcinoma (ccRCC) tumors, in good agreement with inductively coupled mass spectroscopy. Our data suggest that TPL microscopy stands as a powerful tool to understand and quantify the delivery mechanisms of gold nanoparticles, highly relevant to the development of future theranostic medicines.