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.

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.

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.

Optical micro-spectroscopy of single metallic nanoparticles: quantitative extinction and transient resonant four-wave mixing.

We report a wide-field imaging method to rapidly and quantitatively measure the optical extinction cross-section σ(ext) (also polarisation resolved) of a large number of individual gold nanoparticles, for statistically-relevant single particle analysis. We demonstrate a sensitivity of 5 nm(2) in σ(ext), enabling detection of single 5 nm gold nanoparticles with total acquisition times in the 1 min range. Moreover, we have developed an analytical model of the polarisation resolved σ(ext), which enabled us to extract geometrical particle aspect ratios from the measured σ(ext). Using this method, we have characterized a large number of nominally-spherical gold nanoparticles in the 10-100 nm size range. Furthermore, the method provided measurements of in-house fabricated nanoparticle conjugates, allowing distinction of individual dimers from single particles and larger aggregates. The same particle conjugates were investigated correlatively by phase-resolved transient resonant four-wave mixing micro-spectroscopy. A direct comparison of the phase-resolved response between single gold nanoparticles and dimers highlighted the promise of the four-wave mixing technique for sensing applications with dimers as plasmon rulers.

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.

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.

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.

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds.

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug-delivery vehicles and contrast agents in vivo. In the quest for superior photostability and biocompatibility, nanodiamonds are considered one of the best choices due to their unique structural, chemical, mechanical and optical properties. So far, mainly fluorescent nanodiamonds have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centres with stable optical properties. Here, we show that single non-fluorescing nanodiamonds exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp(3) vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and nanodiamond size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of nanodiamonds internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.