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.

Exciton Dephasing by Phonon-Induced Scattering between Bright Exciton States in InP/ZnSe Colloidal Quantum Dots.

Decoherence or dephasing of the exciton is a central characteristic of a quantum dot (QD) that determines the minimum width of the exciton emission line and the purity of indistinguishable photon emission during exciton recombination. Here, we analyze exciton dephasing in colloidal InP/ZnSe QDs using transient four-wave mixing spectroscopy. We obtain a dephasing time of 23 ps at a temperature of 5 K, which agrees with the smallest line width of 50 µeV we measure for the exciton emission of single InP/ZnSe QDs at 5 K. By determining the dephasing time as a function of temperature, we find that exciton decoherence can be described as a phonon-induced, thermally activated process. The deduced activation energy of 0.32 meV corresponds to the small splitting within the nearly isotropic bright exciton triplet of InP/ZnSe QDs, suggesting that the dephasing is dominated by phonon-induced scattering within the bright exciton triplet.

Hybrid Plasmonic Nanostructures for Enhanced Single-Molecule Detection Sensitivity.

Biosensing applications based on fluorescence detection often require single-molecule sensitivity in the presence of strong background signals. Plasmonic nanoantennas are particularly suitable for these tasks, as they can confine and enhance light in volumes far below the diffraction limit. The recently introduced antenna-in-box (AiB) platforms achieved high single-molecule detection sensitivity at high fluorophore concentrations by placing gold nanoantennas in a gold aperture. However, hybrid AiB platforms with alternative aperture materials such as aluminum promise superior performance by providing better background screening. Here, we report on the fabrication and optical characterization of hybrid AiBs made of gold and aluminum for enhanced single-molecule detection sensitivity. We computationally optimize the optical properties of AiBs by controlling their geometry and materials and find that hybrid nanostructures not only improve signal-to-background ratios but also provide additional excitation intensity and fluorescence enhancements. We further establish a two-step electron beam lithography process to fabricate hybrid material AiB arrays with high reproducibility and experimentally validate the higher excitation and emission enhancements of the hybrid nanostructures as compared to their gold counterpart. We foresee that biosensors based on hybrid AiBs will provide improved sensitivity beyond the capabilities of current nanophotonic sensors for a plethora of biosensing applications ranging from multicolor fluorescence detection to label-free vibrational spectroscopy.

Correlative light-electron microscopy using small gold nanoparticles as single probes.

Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.

uFLIM - Unsupervised analysis of FLIM-FRET microscopy data.

Despite their widespread use in cell biology, fluorescence lifetime imaging microscopy (FLIM) data-sets are challenging to analyse, because each spatial position can contain a superposition of multiple fluorescent components. Here, we present a data analysis method employing all information in the available photon budget, as well as being fast. The method, called uFLIM, determines spatial distributions and temporal dynamics of multiple fluorescent components with no prior knowledge. It goes significantly beyond current approaches which either assume the functional dependence of the dynamics, e.g. an exponential decay, or require dynamics to be known, or calibrated. Its efficient non-negative matrix factorization algorithm allows for real-time data processing. We validate in silico that uFLIM is capable to disentangle the spatial distribution and spectral properties of five fluorescing probes, from only two excitation and detection channels and a photon budget of 100 detected photons per pixel. By adapting the method to data exhibiting Förster resonant energy transfer (FRET), we retrieve the spatial and transfer rate distribution of the bound species, without constrains on donor and acceptor dynamics.

Quantitatively linking morphology and optical response of individual silver nanohedra.

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.

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.

Biofunctionalisation of gallium arsenide with neutravidin.

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.

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.

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.

Quantification of the nonlinear susceptibility of the hydrogen and deuterium stretch vibration for biomolecules in coherent Raman micro-spectroscopy.

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.

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.

The optical nanosizer - quantitative size and shape analysis of individual nanoparticles by high-throughput widefield extinction microscopy.

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 nm2 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.

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.

Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist.

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400-580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 µm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm-1. Secondary Harmonic Generation highlights the highly ordered structure of the flagella.

Four-wave-mixing microscopy reveals non-colocalisation between gold nanoparticles and fluorophore conjugates inside cells.

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.

Quantitative optical microspectroscopy, electron microscopy, and modelling of individual silver nanocubes reveal surface compositional changes at the nanoscale.

The optical response of metal nanoparticles is governed by plasmonic resonances, which depend often intricately on the geometry and composition of the particle and its environment. In this work we describe a method and analysis pipeline unravelling these relations at the single nanoparticle level through a quantitative characterization of the optical and structural properties. It is based on correlating electron microscopy with microspectroscopy measurements of the same particle immersed in media of different refractive indices. The optical measurements quantify the magnitude of both the scattering and the absorption cross sections, while the geometry measured in electron microscopy is used for numerical simulations of the cross section spectra accounting for the experimental conditions. We showcase the method on silver nanocubes of nominal 75 nm edge size. The large amount of information afforded by the quantitative cross section spectra and measuring the same particle in two environments, allows us to identify a specific degradation of the cube surface. We find a layer of tarnish, only a few nanometers thick, a fine surface compositional change of the studied system which would be hardly quantifiable otherwise.