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The optical response of metal nanoparticles is governed by plasmonic resonances, which are dictated by the particle morphology. A thorough understanding of the link between morphology and optical response requires quantitatively measuring optical and structural properties of the same particle. Here we present such a study, correlating electron tomography and optical micro-spectroscopy. The optical measurements determine the scattering and absorption cross-section spectra in absolute units, and electron tomography determines the 3D morphology. Numerical simulations of the spectra for the individual particle geometry, and the specific optical set-up used, allow for a quantitative comparison including the cross-section magnitude. Silver nanoparticles produced by photochemically driven colloidal synthesis, including decahedra, tetrahedra and bi-tetrahedra are investigated. A mismatch of measured and simulated spectra is found in some cases when assuming pure silver particles, which is explained by the presence of a few atomic layers of tarnish on the surface, not evident in electron tomography. The presented method tightens the link between particle morphology and optical response, supporting the predictive design of plasmonic nanomaterials.

Quantitative hyperspectral coherent Raman scattering microscopy merges imaging with spectroscopy and utilises quantitative data analysis algorithms to extract physically meaningful chemical components, spectrally and spatially-resolved, with sub-cellular resolution. This label-free non-invasive method has the potential to significantly advance our understanding of the complexity of living multicellular systems. Here, we have applied an in-house developed hyperspectral coherent anti-Stokes Raman scattering (CARS) microscope, combined with a quantitative data analysis pipeline, to imaging living mouse liver organoids as well as fixed mouse brain tissue sections xenografted with glioblastoma cells. We show that the method is capable of discriminating different cellular sub-populations, on the basis of their chemical content which is obtained from an unsupervised analysis, i.e. without prior knowledge. Specifically, in the organoids, we identify sub-populations of cells at different phases in the cell cycle, while in the brain tissue, we distinguish normal tissue from cancer cells, and, notably, tumours derived from transplanted cancer stem cells versus non-stem glioblastoma cells. The ability of the method to identify different sub-populations was validated by correlative fluorescence microscopy using fluorescent protein markers. These examples expand the application portfolio of quantitative chemical imaging by hyperspectral CARS microscopy to multicellular systems of significant biomedical relevance, pointing the way to new opportunities in non-invasive disease diagnostics., Quantitative hyperspectral coherent Raman scattering microscopy merges imaging with spectroscopy and utilises quantitative data analysis algorithms to extract chemical components, spectrally and spatially-resolved, with sub-cellular resolution.

Gold nanoparticles have been researched for many biomedical applications in diagnostics, theranostics, and as drug delivery systems. When conjugated to fluorophores, their interaction with biological cells can be studied in situ and real time using fluorescence microscopy. However, an important question that has remained elusive to answer is whether the fluorophore is a faithful reporter of the nanoparticle location. Here, our recently developed four-wave-mixing optical microscopy is applied to image individual gold nanoparticles and in turn investigate their co-localisation with fluorophores inside cells. Nanoparticles from 10 nm to 40 nm diameter were conjugated to fluorescently-labeled transferrin, for internalisation via clathrin-mediated endocytosis, or to non-targeting fluorescently-labelled antibodies. Human (HeLa) and murine (3T3-L1) cells were imaged at different time points after incubation with these conjugates. Our technique identified that, in most cases, fluorescence originated from unbound fluorophores rather than from fluorophores attached to nanoparticles. Fluorescence detection was also severely limited by photobleaching, quenching and autofluorescence background. Notably, correlative extinction/fluorescence microscopy of individual particles on a glass surface indicated that commercial constructs contain large amounts of unbound fluorophores. These findings highlight the potential problems of data interpretation when reliance is solely placed on the detection of fluorescence within the cell, and are of significant importance in the context of correlative light electron microscopy.

Deuterium labelling is increasingly used in coherent Raman imaging of complex systems, such as biological cells and tissues, to improve chemical specificity. Nevertheless, quantitative coherent Raman susceptibility spectra for deuterated compounds have not been previously reported. Interestingly, it is expected theoretically that –D stretch vibrations have a Raman susceptibility lower than –H stretch vibrations, with the area of their imaginary part scaling with their wavenumber, which is shifted from around 2900 cm−1 for C–H into the silent region around 2100 cm−1 for C–D. Here, we report quantitative measurements of the nonlinear susceptibility of water, succinic acid, oleic acid, linoleic acid and deuterated isoforms. We show that the –D stretch vibration has indeed a lower area, consistent with the frequency reduction due to the doubling of atomic mass from hydrogen to deuterium. This finding elucidates an important trade-off between chemical specificity and signal strength in the adoption of deuterium labelling as an imaging strategy for coherent Raman microscopy.

Gallium arsenide (GaAs) is a promising candidate as a platform for optical biosensing devices due to its enabling optoelectronic properties. However, the biofunctionalisation of the GaAs surface has not received much attention compared to gold, carbon and silicon surfaces. Here we report a study presenting a physicochemical surface characterisation of the GaAs surface along the functionalisation with a high-affinity bioconjugation pair widely explored in the life sciences – biotin and neutravidin. Combined X-ray photoelectron spectroscopy (XPS), wettability measurements and spectroscopic ellipsometry were used for a reliable characterisation of the surface functionalisation process. The results suggest that a film with a thickness lower than 10 nm was formed, with a neutravidin to biotin ratio of 1:25 on the GaAs surface. Reduction of non-specific binding of the protein to the surface was achieved by optimising the protein buffer and rinsing steps. This study shows the feasibility of using GaAs as a platform for specific biomolecular recognition, paving the way to a new generation of optoelectronic biosensors.

Quantifying the optical extinction cross section of a plasmonic nanoparticle has recently emerged as a powerful means to characterize the nanoparticle morphologically, i.e., to determine its size and shape with a precision comparable to electron microscopy while using a simple optical microscope. In this context, a critical piece of information to solve the inverse problem, namely, calculating the particle geometry from the measured cross section, is the material permittivity. For bulk gold, many datasets have been reported in the literature, raising the question of which one is more adequate to describe specific systems at the nanoscale. Another question is how the nanoparticle interface, not present in the bulk material, affects its permittivity. In this work, we have investigated the role of the material permittivities on the morphometric characterization of defect-free ultra-uniform gold nanospheres with diameters of 10 nm and 30 nm, following a quantitative analysis of the polarization- and spectrally-resolved extinction cross section on hundreds of individual nanoparticles. The measured cross sections were fitted using an ellipsoid model. By minimizing the fit error or the variation of the fitted dimensions with color channel selection, the material permittivity dataset and the surface damping parameter g best describing the nanoparticles are found to be the single crystal dataset by Olmon et al. [Phys. Rev. B 86, 235147 (2012)] and g approximate to 1, respectively. The resulting nanoparticle geometries are in good agreement with transmission electron microscopy of the same sample batches, including both 2D projection and tomography.

Lipid phase separation in cellular membranes is thought to play an important role in many biological functions. This has prompted the development of synthetic membranes to study lipid–lipid interactions in vitro, alongside optical microscopy techniques aimed at directly visualizing phase partitioning. In this context, there is a need to overcome the limitations of fluorescence microscopy, where added fluorophores can significantly perturb lipid packing. Raman-based optical imaging is a promising analytical tool for label-free chemically specific microscopy of lipid bilayers. In this work, we demonstrate the application of hyperspectral coherent Raman scattering microscopy combined with a quantitative unsupervised data analysis methodology developed in-house to visualize lipid partitioning in single planar membrane bilayers exhibiting liquid-ordered and liquid-disordered domains. Two home-built instruments were utilized, featuring coherent anti-Stokes Raman scattering and stimulated Raman scattering modalities. Ternary mixtures of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol were used to form phase-separated domains. We show that domains are consistently resolved, both chemically and spatially, in a completely label-free manner. Quantitative Raman susceptibility spectra of the domains are provided alongside their spatially resolved concentration maps.

In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U-2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 104 to 105 molecules. Based on the measured number of eGFP tagged cyclin B1 proteins, knowledge of cyclin B1 dynamics through the cell cycle, and the cell morphology, we identify the stages of cells in the cell cycle. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Nanoparticles are widely utilised for a range of applications, from catalysis to medicine, requiring accurate knowledge of their size and shape. Current techniques for particle characterisation are either not very accurate or time consuming and expensive. Here we demonstrate a rapid and quantitative method for particle analysis based on measuring the polarisation-resolved optical extinction cross-section of hundreds of individual nanoparticles using wide-field microscopy, and determining the particle size and shape from the optical properties. We show measurements on three samples consisting of nominally spherical gold nanoparticles of 20 nm and 30 nm diameter, and gold nanorods of 30 nm length and 10 nm diameter. Nanoparticle sizes and shapes in three dimensions are deduced from the measured optical cross-sections at different wavelengths and light polarisation, by solving the inverse problem, using an ellipsoid model of the particle polarisability in the dipole limit. The sensitivity of the method depends on the experimental noise and the choice of wavelengths. We show an uncertainty down to about 1 nm in mean diameter, and 10% in aspect ratio when using two or three color channels, for a noise of about 50 nm^2 in the measured cross-section. The results are in good agreement with transmission electron microscopy, both 2D projection and tomography, of the same sample batches. Owing to its combination of experimental simplicity, ease of access to statistics over many particles, accuracy, and geometrical particle characterisation in 3D, this 'optical nanosizer' method has the potential to become the technique of choice for quality control in next-generation particle manufacturing.

Coherent anti-Stokes Raman scattering (CARS) implemented as a vibrational micro-spectroscopy modality eradicates the need for potentially perturbative fluorescent labeling while still providing high-resolution, chemically specific images of biological samples. Isotopic substitution of hydrogen atoms with deuterium introduces minimal change to molecular structures and can be coupled with CARS microscopy to increase chemical contrast. Here, we investigate HeLa cells incubated with non-deuterated or deuterium-labeled fatty acids, using an in-house-developed hyperspectral CARS microscope coupled with an unsupervised quantitative data analysis algorithm, to retrieve Raman susceptibility spectra and concentration maps of chemical components in physically meaningful units. We demonstrate that our unsupervised analysis retrieves the susceptibility spectra of the specific fatty acids, both deuterated and non-deuterated, in good agreement with reference Raman spectra measured in pure lipids. Our analysis, using the cell-silent spectral region, achieved excellent chemical specificity despite having no prior knowledge and considering the complex intracellular environment inside cells. The quantitative capabilities of the analysis allowed us to measure the concentration of deuterated and non-deuterated fatty acids stored within cytosolic lipid droplets over a 24 h period. Finally, we explored the potential use of deuterium-labeled lipid droplets for non-invasive cell tracking, demonstrating an effective application of the technique for distinguishing between cells in a mixed population over a 16 h period. These results further demonstrate the chemically specific capabilities of hyperspectral CARS microscopy to characterize and distinguish specific lipid types inside cells using an unbiased quantitative data analysis methodology.

Quantitative differential interference contrast microscopy is demonstrated here as a label-free method, which is able to image and measure the thickness of lipid bilayers with 0.1 nm precision. We investigate the influence of the substrate on the thickness of fluid-phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-supported lipid bilayers and find a thinning of up to 10%, depending on substrate hydrophilicity, local bilayer coverage, and ionic strength of the medium. With fluorescently labeled lipid bilayers, we also observe changes in the bilayer thickness depending on the choice of fluorophore. Furthermore, liquid-ordered domains in bilayers, formed from DOPC, cholesterol, and sphingomyelin, are measured, and the corresponding thickness change between the liquid-ordered and liquid-disordered phases is accurately determined. Again, the thickness difference is found to be dependent on the presence of the fluorophore label, highlighting the need for quantitative label-free techniques.

Mammalian eggs generate most of their ATP by mitochondrial oxidation of pyruvate from the surrounding medium or from fatty acids that are stored as triacylglycerols within lipid droplets. The balance between pyruvate and fatty acid oxidation in generating ATP is not established. We have combined coherent anti-Stokes Raman scattering (CARS) imaging with deuterium labelling of oleic acid to monitor turnover of fatty acids within lipid droplets of living mouse eggs. We found that loss of labelled oleic acid is promoted by pyruvate removal but minimised when β-oxidation is inhibited. Pyruvate removal also causes a significant dispersion of lipid droplets, while inhibition of β-oxidation causes droplet clustering. Live imaging of luciferase or FAD autofluorescence from mitochondria, suggest that inhibition of β-oxidation in mouse eggs only leads to a transient decrease in ATP because there is compensatory uptake of pyruvate into mitochondria. Inhibition of pyruvate uptake followed by β-oxidation caused a similar and successive decline in ATP. Our data suggest that β-oxidation and pyruvate oxidation contribute almost equally to resting ATP production in resting mouse eggs and that reorganisation of lipid droplets occurs in response to metabolic demand.

Many promising techniques proposed to monitor gamete developmental potential and quality are invasive and not realistically useful in clinical practise. Hence, there is increasing interest in the development of non-invasive imaging methods that can be applied to mammalian eggs and early embryos. Recent studies have shown that mammalian oocyte and embryo viability are closely associated with their metabolic profile, relying entirely on mitochondria as a source of ATP. Fatty acids, stored in intracellular lipid droplets, are an important source of ATP. We have recently demonstrated the use of Coherent Anti-stokes Raman Scattering (CARS) microscopy to allow chemically-specific, label-free imaging of lipid droplets, with high three-dimensional spatial resolution. Here, we summarize our main findings when using CARS to examine the number, size, and 3D spatial distribution of lipid droplets in mouse eggs and early embryos. Quantitative analysis showed statistically significant differences during oocyte maturation and early embryo development. Notably, CARS imaging did not compromise maturation or development. In mouse oocytes that had been subjected to alterations in mitochondrial metabolism we found that the spatial distribution pattern of lipid droplets was also altered. In addition, differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids were detected using CARS hyperspectral imaging. We also imaged bovine oocytes, and found that lipid droplets appear to be larger and with less spatial aggregation than in mouse oocytes, possibly reflecting the fact that different species metabolise lipids differently. These data suggest that CARS microscopy is a promising non-invasive technique for assessing specific aspects of the metabolic profile of living mammalian eggs and early embryos, which could be potentially linked to their quality and viability.

Microscopy-based high-content and high-throughput analysis of cellular systems plays a central role in drug discovery. However, for contrast and specificity, the majority of assays require a fluorescent readout which always comes with the risk of alteration of the true biological conditions. In this work, we demonstrate a label-free imaging platform which combines chemically specific hyperspectral coherent anti-Stokes Raman scattering microscopy with sparse sampling and Bessel beam illumination. This enabled us to screen multiwell plates at high speed, while retaining the high-content chemical analysis of hyperspectral imaging. To demonstrate the practical applicability of the method we addressed a critical side effect in drug screens, namely, drug-induced lipid storage within hepatic tissue. We screened 15 combinations of drugs and neutral lipids added to human HepG2 liver cells and developed a high-content quantitative data analysis pipeline which extracted the spectra and spatial distributions of lipid and protein components. We then used their combination to train a support vector machine discriminative algorithm. Classification of the drug responses in terms of phospholipidosis versus steatosis was achieved in a completely label-free assay.

© 2018 American Chemical Society. Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.

Non-negative factorization analysis applied to spontaneous Brillouin microscopy maps of amyloid-beta plaques in a transgenic mouse model enables to resolve spatially distinct components with specific mechanical properties., A recent investigation on the architecture and chemical composition of amyloid-β (Aβ) plaques in ex vivo histological sections of an Aβ-overexpressing transgenic mouse hippocampus has shed light on the infrared light signature of cell-activation related biomarkers of Alzheimer's disease. A correlation was highlighted between the biomechanical properties detected by Brillouin microscopy and the molecular make-up of Aβ plaques provided by FTIR spectroscopic imaging and Raman microscopy (with correlative immunofluorescence imaging) in this animal model of the disease. In the Brillouin spectra of heterogeneous materials such as biomedical samples, peaks are likely the result of multiple contributions, more or less overlaid on a spatial and spectral scale. The ability to disentangle these contributions is very important as it may give access to discrete components that would otherwise be buried within the Brillouin peak envelope. Here, we applied an unsupervised non-negative matrix factorization method to analyse the spontaneous Brillouin microscopy maps of Aβ plaques in transgenic mouse hippocampal sections. The method has already been proven successful in decomposing chemical images and is applied here for the first time to acoustic maps acquired with a Fabry–Perot Brillouin microscope. We extracted and visualised a decrease in tissue rigidity from the core through to the periphery of the plaque, with spatially distinct components that we assigned to specific entities. This work demonstrates that it is possible to reveal the structure and mechanical properties of Aβ plaques, with details visualized by the projection of the mechanical contrast into a few relevant channels.

We have investigated the ability of dual-frequency Coherent Antistokes Raman Scattering (D-CARS) micro-spectroscopy, based on femtosecond pulses (100 fs or 5 fs) spectrally focussed by glass dispersion, to distinguish the chemical composition of micron-sized lipid droplets consisting of different triglycerides types (poly-unsaturated glyceryl trilinolenate, mono-unsaturated glyceryl trioleate and saturated glyceryl tricaprylate and glyceryl tristearate) in a rapid and label-free way. A systematic comparison of Raman spectra with CARS and D-CARS spectra was used to identify D-CARS spectral signatures which distinguish the disordered poly-unsaturated lipids from the more ordered saturated ones both in the CH-stretch vibration region and in the fingerprint region, without the need for lengthy CARS multiplex acquisition and analysis. D-CARS images of the lipid droplets at few selected wavenumbers clearly resolved the lipid composition differences, and exemplify the potential of this technique for label-free chemically selective rapid imaging of cytosolic lipid droplets in living cells.

We demonstrate a technique for simultaneous detection of coherent anti-Stokes Raman scattering (CARS) at four vibrational frequencies, using simple passive optical elements and without spectrally resolved detection. The technique is based on pump and Stokes femtosecond pulses selectively exciting vibrational resonances through spectral focusing. By replicating the pump and Stokes pair into four pairs, each traveling through appropriate glass elements, we simultaneously excite four different vibrational frequencies. The resulting CARS is a periodic train of intensities detected by a single photomultiplier and frequency analyzed to retrieve its Fourier coefficients. We demonstrate detection of methanol and ethanol mixtures in water and quantitative determination of their concentration owing to the improved chemical selectivity of this quadruplex CARS scheme.

The surface conformation and accessibility of oligonucleotides within arrays are two key parameters that affect the utility of immobilized nucleic acids in sensor technologies. In this work, a novel combination of analytical techniques was used to compare two methods for DNA immobilization on glass. The aim of the study was to identify a method that generated a high surface density of hybridization-accessible oligonucleotides in a true planar monolayer. The first method based on direct coupling of silanized DNA to the glass surface showed a high immobilization density of 0.013 molecules/nm2 but low surface accessibility, as shown by the hybridization measurements (or =15%). The second method, based on the biotin-streptavidin interaction, generated a high immobilization density (0.02 molecules/nm2) and high surface accessibility (90%). Atomic force microscopy and X-ray photoelectron spectroscopy indicated that both methods achieved uniform surfaces. Using the biotin-streptavidin system, the intermolecular distance between the hybridized molecules could be tightly controlled by titrating biotinylated complementary and noncomplementary oligonucleotides.

Using a differential transmission pump-probe experiment in heterodyne detection, the ultrafast gain and refractive-index dynamics of the ground-state transition in InAs/GaAs quantum- dot amplifiers emitting near 1.3 mum at working condition, that is at room temperature and under electrical injection were measured. An ultrafast gain recovery on a subpicosecond time scale is observed at high electrical injection indicating fast carrier relaxation into the dot ground state, which is appealing for high-speed applications with these devices. Comparing p-doped and undoped devices of otherwise identical structure and operating at the same gain, a faster absorption recovery but a slower gain dynamics in p-doped amplifiers was observed. This finding should help in elucidating the role of p-doping in the design of QD-based devices with high-speed performances.

We measure the dephasing time of the first optically-active excited-state excitonic transition in strongly confined InGaAs quantum dots using transient four-wave mixing. The optically-driven excited-state polarization shows a bi-exponential decay with a significant fraction of the probed excited states exhibiting a very long dephasing time, in the nanosecond range at 10 K. The full time-dependent four-wave mixing polarization is microscopically calculated by taking into account both virtual and real acoustic phonon-assisted transitions. The bi-exponential decay is qualitatively explained by a model of two-bright excitonic excited states non-degenerate in the absence of in-plane cylindrical symmetry. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

In this paper, the present status of the experimental study of the optical properties of biexcitons in semiconductor microcavities is reviewed. In particular, a detailed investigation of a polariton–biexciton transition in a high-quality single quantum well GaAs/AlGaAs microcavity is reported. The binding energy and the coherent dynamics of the biexciton state are measured at low temperature using differential reflectivity and transient four-wave mixing spectroscopy, respectively. It is found that the biexciton binding energy is not strongly influenced by the exciton–photon coupling in the microcavity, even if the vacuum Rabi splitting exceeds the biexciton binding energy. However, the presence of a longitudinal built-in electric field that results in a Stark effect slightly reducing the binding energy compared to the value measured on a reference bare quantum well is experimentally pointed out and compared with calculations. Additionally, the polarization decay of the transition from the crystal ground state to the biexciton is measured and is shown to be larger by approximately a factor of two compared to the value measured on the reference quantum well.

We present measurements of the dephasing time of biexcitonic transitions in self-organized InGaAs quantum dots in the temperature range from 10 K to 70 K. We use an heterodyne four-wave mixing technique that allows direct measurement of the dephasing time in an inhomogeneously broadened quantum-dot ensemble. By applying an electrical injection, we progressively increase the number of carriers in the quantum dots and investigate the passage from a four-wave mixing response dominated by the one-exciton transition to a response dominated by biexcitonic and multiexcitonic transitions. The temperature dependence of the homogeneous broadening of the biexciton-to-exciton transition is extrapolated to zero injection current and its physical origin in terms of radiative recombination and phonon interactions is discussed.

Time-resolved four-wave mixing in strongly confined and electrically pumped InGaAs quantum dots is measured at different temperatures. Without electrical injection, the dephasing time of the dot ground state transition is lifetime-limited and of several hundred picoseconds at low temperature. Under electrical injection, the sum of the contributions from empty and populated dots with a relative phase shift of π is evidenced near transparency.

The ultrafast gain and index dynamics in a set of InAs-InGaAs-GaAs quantum-dot (QD) amplifiers are measured at room temperature with femtosecond resolution. The role of spectral hole-burning (SHB) and carrier heating (CH) in the recovery of gain compression is investigated in detail. An ultrafast recovery of the spectral hole within /spl sim/100 fs is measured, comparable to bulk and quantum-well amplifiers, which is contradicting a carrier relaxation bottleneck in electrically pumped QD devices. The CH dynamics in the QD is quantitatively compared with results on an InGaAsP bulk amplifier. Reduced CH for both gain and refractive index dynamics of the QD devices is found, which is a promising prerequisite for high-speed applications. This reduction is attributed to reduced free-carrier absorption-induced heating caused by the small carrier density necessary to provide amplification in these low-dimensional systems.

Summary form only given. Semiconductor quantum dots (QDs) are receiving increasing attention for fundamental studies on zero-dimensional confinement and for device applications. Quantum-dot lasers are expected to show superior performances, like high material gain, low and temperature-independent threshold current and chirp-free operation, due to the delta-like density of states (DOS). We have measured the dephasing time at room temperature of InAs QDs embedded in a waveguide to estimate the lower limit for the energy-broadening of the DOS given by the homogeneous linewidth. The sample consists of 3 stacked layers of InAs-InGaAs-GaAs quantum dots.

Photoluminescence excitation (PLE) measurements have been performed in several GaAs-based structures by monitoring the electron-carbon acceptor and the exciton bound to carbon recombinations. In both cases we show that the separate capture of free carriers makes the main contribution to the electron-carbon transition and to the formation of the bound exciton. A dip is indeed observed in the PLE spectra at the energy of the free exciton. The contribution of the bound exciton relaxation to the two-hole transition is pointed out.

The photoluminescence of GaAs / AlAs source multiple quantum wells structures under optical ps excitation is investigated for carrier densities in the range 1018−4 × 1019 cm−3 with frequency and time-resolved spectroscopic techniques. The measurements give a direct evidence of the occurrence in the sample of carrier heating. This energy up-conversion gives rise to photoluminescence from the states near the Fermi level whose intensity and time evolution depend on the carrier density in a strongly non-linear way. The observed behaviour can be explained introducing in the carrier dynamics an up-conversion mechanism due to Auger-like processes.

Using a phase-sensitive degenerate four-wave mixing technique in heterodyne detection we measured the ultrafast changes of the complex polarizability in single gold nanoparticles at the surface plasmon resonance. Two components in the nanoparticle nonlinear response are distinguished, depending on the linear polarization direction of pump and probe pulses and particle geometry. One component is quantitatively modeled as the variation of the complex dielectric constant induced by the initial increase in the electron gas temperature following the absorption of the pump pulse, and the subsequent electron thermalization with the lattice and the surrounding. The dependence of the measured four-wave mixing on the probe field polarization direction is well reproduced by including a deviation from spherical symmetry in the nanoparticle dielectric constant. The second component manifests as a significant increase in the measured nonlinearity when the probe field is co-polarized with the pump field at pump–probe time overlap. We attribute this component to the nonlinear response from coherent surface plasmons before dephasing into single electron excitations.

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.

In this work, we report a method to acquire and analyze hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy images of organic materials and biological samples resulting in an unbiased quantitative chemical analysis. The method employs singular value decomposition on the square root of the CARS intensity, providing an automatic determination of the components above noise, which are retained. Complex CARS susceptibility spectra, which are linear in the chemical composition, are retrieved from the CARS intensity spectra using the causality of the susceptibility by two methods, and their performance is evaluated by comparison with Raman spectra. We use non-negative matrix factorization applied to the imaginary part and the nonresonant real part of the susceptibility with an additional concentration constraint to obtain absolute susceptibility spectra of independently varying chemical components and their absolute concentration. We demonstrate the ability of the method to provide quantitative chemical analysis on known lipid mixtures. We then show the relevance of the method by imaging lipid-rich stem-cell-derived mouse adipocytes as well as differentiated embryonic stem cells with a low density of lipids. We retrieve and visualize the most significant chemical components with spectra given by water, lipid, and proteins segmenting the image into the cell surrounding, lipid droplets, cytosol, and the nucleus, and we reveal the chemical structure of the cells, with details visualized by the projection of the chemical contrast into a few relevant channels.

Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging tool for cell biologists to examine live cell dynamics with chemical specificity in a label-free, noninvasive way. CARS is a multiphoton process offering intrinsic three-dimensional submicron resolution, where the image contrast is obtained from light inelastically scattered by the vibrations of endogenous chemical bonds. CARS is particularly well suited to study lipid biology, since the CARS signal of localized lipids (exhibiting a large amount of identical bonds in the focal volume) is very strong. Conversely, photostable, lipid-specific markers for fluorescence microscopy are difficult to produce and the process of labeling often affects lipid localization and function, making imaging lipids in live cells challenging, and accurate quantification often impossible. Here, we describe in detail the principles behind our experimental setup for performing CARS microscopy of lipid droplets on live cells. Since typical vibrational resonances in liquid have coherence times in the picosecond range, CARS is preferably implemented with picosecond lasers which are however expensive and less efficient than femtosecond lasers, which could also be used for other multiphoton techniques such as two-photon fluorescence. In our setup, we show that femtosecond lasers can be spectrally focused in a simple, alignment insensitive, and cost-effective way to achieve a vibrational excitation similar to picosecond lasers. This opens the way to integrate CARS and two-photon fluorescence in a single multimodal instrument for its widespread application. We also describe our dual frequency CARS system which eliminates the nonresonant CARS background offering superior sensitivity and image contrast.

Interactions between biomolecules are an important feature of biological systems and understanding these interactions is a key goal in biochemical studies. Using conventional techniques, such as surface plasmon resonance and isothermal titration calorimetry, the determination of the binding constants requires a significant amount of time and resources to produce and purify sufficient quantities of biomolecules in order to measure the affinity of biological interactions. Using DNA hybridization, we have demonstrated a new technique based on the use of nanotethers and time-resolved Forster resonance energy transfer (FRET) that significantly reduces the amount of material required to carry out quantitative binding assays. Test biomolecules were colocalized and attached to a surface using DNA tethers constructed from overlapping oligonucleotides. The length of the tethers defines the concentration of the tethered biomolecule. Effective end concentrations ranging from 56 nM to 3.8 μM were demonstrated. The use of variable length tethers may have wider applications in the quantitative measurement of affinity binding parameters.

Using a sensitive transient four-wave mixing technique in heterodyne detection, we measured the ground-state excitonic dephasing in PbS colloidal quantum dots of 4–6-nm diameter in the temperature range from 5 to 100 K. We observe an ultrafast (

We provide direct evidence that the macroscopic response of the gain dynamics in electrically-pumped In- GaAs/GaAs quantum dots is a superposition of intradot relaxation dynamics from microstates with multiple discrete carrier numbers. The gain recovery in the presence of an optical pre-pump fully depleting the ground-state gain is measured to be faster than without pre-pump. This effect, opposite to expectations from rate equations with mean-field carrier distributions, is due to a conditional gain recovery in which microstates with slow internal dynamics are suppressed by the pre-pump. The effect is evident at 15K and still observable at 300 K, beneficial for high-speed optical signal processing.

We present the rime-resolved optical characterization of InAs/InGaAs self-assembled quantum dots emitting at 1.3 mu m at room temperature. The photoluminescence decay time varies from 1.2 (5 K) to 1.8 ns (293 K). Evidence of thermalization among dots is seen in both continuous-wave and time-resolved spectra around 150 K. A short rise time of 10+/-2 ps is measured. indicating a fast capture and relaxation of carriers inside the dots. (C) 2000 American Institute of Physics.

We demonstrate coherent anti-Stokes Raman scattering (CARS) microspectroscopy using a single Ti:sapphire laser oscillator and simple passive optical elements. Spectral selection by dichroic mirrors and linear chirping by glass elements creates a vibrational excitation tuneable over a large spectral range ( ∼ 800–2200 cm−1) with adjustable spectral resolution ( ∼ 10–100 cm−1). We furthermore demonstrate the applicability of differential CARS, enhancing the chemical selectivity, with the proposed single-laser configuration.

We have compared the gain dynamics of the ground state excitonic transition between undoped and p-doped electrically-pumped InGaAs quantum-dot optical amplifiers, for temperatures from 300K to 20K. A pump-probe differential transmission technique in heterodyne detection with sub-picosecond time resolution was used. The comparison shows that in the gain regime at high temperatures the recovery dynamics of the p-doped sample is slower than in the undoped device operating at the same modal gain, due to a reduced electron reservoir in the excited states. Conversely, at 20K the initial gain dynamics is faster in the p-doped device due to hole-hole scattering. © 2009 SPIE.

Ultrafast gain dynamics of the ground-state transition are measured in electrically pumped InAs/GaAs quantum-dot amplifiers emitting near 1.3 mu m at room temperature. Gain recovery on a subpicosecond time scale occurs at high electrical injection. However, when comparing p-doped and undoped devices fabricated under identical conditions and operating at the same gain, faster absorption recovery but slower gain dynamics are observed in p-doped amplifiers. The slower gain dynamics is attributed to a reduced reservoir of excited-state electrons in p-doped quantum-dot devices, which limits the recovery of the electron ground-state occupation mediated by intradot carrier-carrier scattering

In this paper , we measure the ultrafast gain and index dynamics of the QD ground state transition in p-doped electrically-pumped InGaAs QD optical amplifiers emitting near 1.3 mum at room temperature. A pump-probe differential transmission technique in heterodyne detection with sub-picosecond time resolution was used. The refractive index dynamics also showed noticeable differences between p-doped and undoped samples. From these data a transient and time-integrated linewidth enhancement factor has been deduced.

This study presents an overview of recent measurements of the dephasing time and gain recovery dynamics in InGaAs/GaAs QD material and devices. Results indicate that the ultrafast gain recovery on a subpicosecond time scale measured at room temperature is related to the presence of several carriers in the excited states of the QD, i.e. to a multiexciton state with several available channels for relaxation.

The dephasing time in semiconductor quantum dots and quantum-dot molecules is measured using a sensitive four-wave mixing heterodyne technique. We find a dephasing time of several hundred picoseconds at low temperature in the ground-state transition of strongly-confined InGaAs quantum dots, approaching the radiative-lifetime limit. Between 7 K and 100 K the polarization decay has two distinct components resulting in a non-Lorentzian lineshape with a zero-phonon line and a broad band from elastic exciton-acoustic phonon interactions. On a series of InAs/GaAs quantum-dot molecules having different interdot barrier thicknesses a systematic dependence of the dephasing dynamics on the barrier thickness is observed. The results show how the quantum mechanical coupling of the electronic wavefunctions in the molecules affects both the exciton radiative lifetime and the exciton-acoustic phonon interaction.

We report the experimental observation and the theoretical modeling of self-induced-transparency signatures such as nonlinear transmission, pulse retardation and reshaping, for subpicosecond pulse propagation in a 2-mm-long InGaAs quantum-dot ridge waveguide in resonance with the excitonic ground-state transition at 10 K. The measurements were obtained by using a cross-correlation frequency-resolved optical gating technique which allows us to retrieve the field amplitude of the propagating pulses.

We have measured the exciton dephasing time in InAs=GaAs quantum dot molecules having different interdot barrier thicknesses in the temperature range from 5 to 60 K, using a highly sensitive four-wave mixing heterodyne technique. At 5 K dephasing times of several hundred picoseconds are found.\ud Moreover, a systematic dependence of the dephasing dynamics on the barrier thickness is observed.\ud These results show how the quantum-mechanical coupling of the electronic wave functions in the molecules affects both the exciton radiative lifetime and the exciton-acoustic phonon interaction.

Summary form only given. Semiconductor lasers with an active medium containing zero-dimensional structures are expected to show superior performance, like high material gain and low threshold current. In this work we have measured the transmission properties and the carrier dynamics of InAs/GaAs QD amplifiers at room temperature. The samples consist of 3 stacked layers of InAs/InGaAs/GaAs quantum dots.

The coherent response of excitons in semiconductor nanostructures, as measured in four wave mixing (FWM) experiments, depends strongly on the inhomogeneous broadening of the exciton transition. We investigate GaAs-AlGaAs single quantum wells (SQW) of 4 nm to 25 nm well width. Two main mechanisms are found to be important. First, the excitation-induced dephasing FWM signal (EID), which leads to a strong dependence of the signal on the angle between the linear input polarizations. The presence of the EID in the mainly homogeneously broadened sample (25 nm) is shown by the beating between EID and two-photon coherence (TPC) at the exciton for negative delay and co-linear polarized excitation. It vanishes for cross-polarized excitation, thus excluding the importance of local-field effects.

We present temperature-dependent measurements of the dephasing time in the ground-state transition of strongly-confined InGaAs quantum dots, using a highly sensitive four-wave mixing technique. At low temperature we measure a dephasing time of several hundred picoseconds. Between 7 and 100 K the polarization decay has two distinct components resulting in a non-Lorentzian lineshape with a sharp zero-phonon line and a broad band from elastic exciton-acoustic phonon interactions. We also explore the dephasing time beyond the one exciton occupation, by electrically injecting carriers. Electrical injection into the barrier region results in a dominantly pure dephasing of the excitonic ground-state transition. Once the injected carriers have filled the electronic ground state, additional filling of the excited states creates multiexcitons that show a fast dephasing due to population relaxation.

We have achieved a record high ratio (19) of the Rabi splitting (3.6 meV) to the polariton linewidth (190 mu eV), in a semiconductor lambda microcavity with a single 25 nm GaAs quantum well at the antinode. The narrow polariton lines are obtained with a special cavity design which reduces the exciton broadening due to scattering with free charges and has a very low spatial gradient of the cavity resonance energy. Since the static quantum-well disorder is very small, the polariton broadening is dominantly homogeneous. Still, the measured linewidths close to zero detuning cannot be correctly predicted using the linewidth averaging model. (C) 2000 American Institute of Physics.