Low-energy electron microscopy intensity-voltage data - Factorization, sparse sampling and classification.

Low-energy electron microscopy (LEEM) taken as intensity-voltage (I-V) curves provides hyperspectral images of surfaces, which can be used to identify the surface type, but are difficult to analyse. Here, we demonstrate the use of an algorithm for factorizing the data into spectra and concentrations of characteristic components (FSC3 ) for identifying distinct physical surface phases. Importantly, FSC3 is an unsupervised and fast algorithm. As example data we use experiments on the growth of praseodymium oxide or ruthenium oxide on ruthenium single crystal substrates, both featuring a complex distribution of coexisting surface components, varying in both chemical composition and crystallographic structure. With the factorization result a sparse sampling method is demonstrated, reducing the measurement time by 1-2 orders of magnitude, relevant for dynamic surface studies. The FSC3 concentrations are providing the features for a support vector machine-based supervised classification of the surface types. Here, specific surface regions which have been identified structurally, via their diffraction pattern, as well as chemically by complementary spectro-microscopic techniques, are used as training sets. A reliable classification is demonstrated on both example LEEM I-V data sets.

Optical absorption and dichroism of single melanin nanoparticles.

Melanin nanoparticles (NPs) have important biological functions including photoprotection and colouration, and artificial melanin-like NPs are relevant for catalysis, drug delivery, diagnosis and therapy. Despite their importance, the optical properties of single melanin NPs have not been measured. We combine quantitative differential interference contrast (qDIC) and extinction microscopy to characterise the optical properties of single NPs, both naturally sourced from cuttlefish ink, as well as synthetic NPs using polydopamine (PDA) and L-3,4-dihydroxyphenylalanine (L-DOPA). Combining qDIC with extinction, we determine the absorption index of individual NPs. We find that on average the natural melanin NPs have a higher absorption index than the artificial melanin NPs. From the analysis of the polarisation-dependent NP extinction, the NP aspect ratio is determined, with mean values at 405 nm wavelength in agreement with transmission electron microscopy. At longer wavelengths, we observe an additional optical anisotropy which is attributed to dichroism by structural ordering of the melanin. Our quantitative analysis yields a dichroism of 2-10% of the absorption index, increasing with wavelength from 455 nm to 660 nm for L-DOPA and PDA. Such an in-depth quantification of the optical properties of single melanin NPs is important for the design and future application of these ubiquitous bionanomaterials.

Quantum Mollow Quadruplet in Nonlinear Cavity QED.

We develop an exact analytical approach to the optical response of a two-level system coupled to a microcavity for arbitrary excitation strengths. The response is determined in terms of the complex amplitudes of transitions between the rungs of the Jaynes-Cummings ladder, explicitly isolating nonlinearities of different orders. Increasing the pulse area of the excitation field, we demonstrate the formation of a quantum Mollow quadruplet (QMQ), quantizing the semiclassical Mollow triplet into a coherent superposition of a large number of transitions between rungs of the ladder, with inner and outer doublets of the QMQ formed by densely lying inner and outer quantum transitions between the split rungs. Remarkably, a closed-form analytic approximation for the QMQ of any order of nonlinearity is found in the high-field low-damping limit.

Sizing individual dielectric nanoparticles with quantitative differential interference contrast microscopy.

We report a method to measure the size of single dielectric nanoparticles with high accuracy and precision using quantitative differential interference contrast (DIC) microscopy. Dielectric nanoparticles are detected optically by the conversion of the optical phase change into an intensity change using DIC. Phase images of individual nanoparticles were retrieved from DIC by Wiener filtering, and a quantitative methodology to extract nanoparticle sizes was developed. Using polystyrene beads of 100 nm radius as size standard, we show that the method determines this radius within a few nm accuracy. The smallest detectable polystyrene bead is limited by background and shot-noise, which depend on acquisition and analysis parameters, including the objective numerical aperture, the DIC phase offset, and the refractive index contrast between particles and their surrounding. Measurements on small beads of 15 nm nominal radius are shown, and a sensitivity limit potentially reaching down to 1.8 nm radius was inferred. As application example, individual nanodiamonds with nominal sizes below 50 nm were measured, and were found to have a nearly exponential size distribution with 28 nm mean value. Considering the importance of dielectric nanoparticles in many fields, from naturally occurring virions to polluting nanoplastics, the proposed method could offer a powerful quantitative tool for nanoparticle analysis, combining accuracy, sensitivity and high-throughput with widely available and easy-to-use DIC microscopy.

Dynamic label-free imaging of lipid droplets and their link to fatty acid and pyruvate oxidation in mouse eggs.

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 ß-oxidation is inhibited. Pyruvate removal also causes a significant dispersion of lipid droplets, while inhibition of ß-oxidation causes droplet clustering. Live imaging of luciferase or FAD autofluorescence from mitochondria, suggest that inhibition of ß-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 ß-oxidation caused a similar and successive decline in ATP. Our data suggest that ß-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.

A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum.

Exposure of mouse oocytes to saturated fatty acids (FAs) such as palmitic acid (PA) has been shown to increase lipid content and cause an endoplasmic reticulum (ER) stress response and changes in the mitochondrial redox state. PA can also disrupt Ca2+ stores in other cell types. The links between these intracellular changes, or whether they are prevented by mono-unsaturated FAs such as oleic acid (OA), is unclear. Here, we have investigated the effects of FAs on mouse oocytes, that are maturated in vitro, using coherent anti-Stokes Raman scattering and two-photon fluorescence microscopy. When oocytes were matured in the presence of PA, there were changes in the aggregation pattern and size of lipid droplets that were mitigated by co-incubation in OA. Maturation in PA alone also caused a distinctive disruption of the ER structure. This effect was prevented by incubation of OA with PA. In contrast, maturation of mouse oocytes in medium containing PA was not associated with any significant change in the redox state of mitochondria or the Ca2+ content of intracellular stores. These data suggest that a primary effect of saturated FAs such as PA on oocytes is to disrupt the structure of the ER and this is not due to an effect on the mitochondria or Ca2+ stores.

Identifying subpopulations in multicellular systems by quantitative chemical imaging using label-free hyperspectral CARS microscopy.

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 morphometric analysis of single gold nanoparticles by optical extinction microscopy: Material permittivity and surface damping effects.

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 ≈ 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.

Hyperspectral CARS microscopy and quantitative unsupervised analysis of deuterated and non-deuterated fatty acid storage in human cells.

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 Label-Free Imaging of Lipid Domains in Single Bilayers by Hyperspectral Coherent Raman Scattering.

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.

Quantitative Imaging of B1 Cyclin Expression Across the Cell Cycle Using Green Fluorescent Protein Tagging and Epifluorescence.

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.

Label-Free Volumetric Quantitative Imaging of the Human Somatic Cell Division by Hyperspectral Coherent Anti-Stokes Raman Scattering.

Quantifying the chemical composition of unstained intact tissue and cellular samples with high spatio-temporal resolution in three dimensions would provide a step change in cell and tissue analytics critical to progress the field of cell biology. Label-free optical microscopy offers the required resolution and noninvasiveness, yet quantitative imaging with chemical specificity is a challenging endeavor. In this work, we show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy can be used to provide quantitative volumetric imaging of human osteosarcoma cells at various stages through cell division, a fundamental component of the cell cycle progress resulting in the segregation of cellular content to produce two progeny. We have developed and applied a quantitative data analysis method to produce volumetric three-dimensional images of the chemical composition of the dividing cell in terms of water, proteins, DNAP (a mixture of proteins and DNA, similar to chromatin), and lipids. We then used these images to determine the dry masses of the corresponding organic components. The attribution of proteins and DNAP components was validated using specific well-characterized fluorescent probes, by comparison with correlative two-photon fluorescence microscopy of DNA and mitochondria. Furthermore, we map the same chemical components under perturbed conditions, employing a drug that interferes directly with cell division (Taxol), showing its influence on cell organization and the masses of proteins, DNAP, and lipids.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos.

Lipid Bilayer Thickness Measured by Quantitative DIC Reveals Phase Transitions and Effects of Substrate Hydrophilicity.

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.

Long Exciton Dephasing Time and Coherent Phonon Coupling in CsPbBr2Cl Perovskite Nanocrystals.

Fully inorganic cesium lead halide perovskite nanocrystals (NCs) have shown to exhibit outstanding optical properties such as wide spectral tunability, high quantum yield, high oscillator strength as well as blinking-free single photon emission, and low spectral diffusion. Here, we report measurements of the coherent and incoherent exciton dynamics on the 100 fs to 10 ns time scale, determining dephasing and density decay rates in these NCs. The experiments are performed on CsPbBr2Cl NCs using transient resonant three-pulse four-wave mixing (FWM) in heterodyne detection at temperatures ranging from 5 to 50 K. We found a low-temperature exciton dephasing time of 24.5 ± 1.0 ps, inferred from the decay of the photon-echo amplitude at 5 K, corresponding to a homogeneous line width (fwhm) of 54 ± 5 µeV. Furthermore, oscillations in the photon-echo signal on a picosecond time scale are observed and attributed to coherent coupling of the exciton to a quantized phonon mode with 3.45 meV energy.

Bessel-Beam Hyperspectral CARS Microscopy with Sparse Sampling: Enabling High-Content High-Throughput Label-Free Quantitative Chemical Imaging.

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.

Realizing the classical XY Hamiltonian in polariton simulators.

The vast majority of real-life optimization problems with a large number of degrees of freedom are intractable by classical computers, since their complexity grows exponentially fast with the number of variables. Many of these problems can be mapped into classical spin models, such as the Ising, the XY or the Heisenberg models, so that optimization problems are reduced to finding the global minimum of spin models. Here, we propose and investigate the potential of polariton graphs as an efficient analogue simulator for finding the global minimum of the XY model. By imprinting polariton condensate lattices of bespoke geometries we show that we can engineer various coupling strengths between the lattice sites and read out the result of the global minimization through the relative phases. Besides solving optimization problems, polariton graphs can simulate a large variety of systems undergoing the U(1) symmetry-breaking transition. We realize various magnetic phases, such as ferromagnetic, anti-ferromagnetic, and frustrated spin configurations on a linear chain, the unit cells of square and triangular lattices, a disordered graph, and demonstrate the potential for size scalability on an extended square lattice of 45 coherently coupled polariton condensates. Our results provide a route to study unconventional superfluids, spin liquids, Berezinskii-Kosterlitz-Thouless phase transition, and classical magnetism, among the many systems that are described by the XY Hamiltonian.

Hyperspectral analysis applied to micro-Brillouin maps of amyloid-beta plaques in Alzheimer's disease brains.

A recent investigation on the architecture and chemical composition of amyloid-ß (Aß) plaques in ex vivo histological sections of an Aß-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 Aß 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 Aß 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 Aß plaques, with details visualized by the projection of the mechanical contrast into a few relevant channels.

Radiatively Limited Dephasing and Exciton Dynamics in MoSe2 Monolayers Revealed with Four-Wave Mixing Microscopy.

By implementing four-wave mixing (FWM) microspectroscopy, we measure coherence and population dynamics of the exciton transitions in monolayers of MoSe2. We reveal their dephasing times T2 and radiative lifetime T1 in a subpicosecond (ps) range, approaching T2 = 2T1 and thus indicating radiatively limited dephasing at a temperature of 6 K. We elucidate the dephasing mechanisms by varying the temperature and by probing various locations on the flake exhibiting a different local disorder. At the nanosecond range, we observe the residual FWM produced by the incoherent excitons, which initially disperse toward the dark states but then relax back to the optically active states within the light cone. By introducing polarization-resolved excitation, we infer intervalley exciton dynamics, revealing an initial polarization degree of around 30%, constant during the initial subpicosecond decay, followed by the depolarization on a picosecond time scale. The FWM hyperspectral imaging reveals the doped and undoped areas of the sample, allowing us to investigate the neutral exciton, the charged one, or both transitions at the same time. In the latter, we observe the exciton-trion beating in the coherence evolution indicating their coherent coupling.

Quantitative Spatiotemporal Chemical Profiling of Individual Lipid Droplets by Hyperspectral CARS Microscopy in Living Human Adipose-Derived Stem Cells.

There is increasing evidence showing that cytosolic lipid droplets, present in all eukaryotic cells, play a key role in many cellular functions. Yet their composition at the individual droplet level and how it evolves over time in living cells is essentially unknown due to the lack of suitable quantitative nondestructive measurement techniques. In this work, we demonstrate the ability of label-free hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy, together with a quantitative image analysis algorithm developed by us, to quantify the lipid type and content in vol/vol concentration units of individual lipid droplets in living human adipose-derived stem cells during differentiation over 9 days in media supplemented with different fatty acids. Specifically, we investigated the addition of the polyunsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing monounsaturated fatty acids). We observe a heterogeneous uptake which is droplet-size dependent, time dependent, and lipid dependent. Cells grown in linoleic-acid-supplemented medium show the largest distribution of lipid content across different droplets at all times during differentiation. When analyzing the average lipid content, we find that adding linoleic or alpha-linolenic fatty acids at day 0 results in uptake of the new lipid components with an exponential time constant of 22 ± 2 h. Conversely, switching lipids at day 3 results in an exponential time constant of 60 ± 5 h. These are unprecedented findings, exemplifying that the quantitative imaging method demonstrated here could open a radically new way of studying and understanding cytosolic lipid droplets in living cells.