Hyperspectral Dark-field Microscopy Research Articles

Hyperspectral dark-field optical microscopy correlated to atomic force microscopy for the analysis of single plasmonic nanoparticles: tutorial

Plasmonic nanostructures are actively investigated for their optical properties and for a wide range of applications in nanophotonics, biosensing, photocatalysis, hot carrier physics, and advanced cancer therapies. The localized surface plasmon resonance (LSPR) can be excited in gold or silver nanoparticles or in more complex nanostructures and gives rise to a wide range of unique optical properties. It is often critical to be able to localize individual plasmonic nanoparticles and simultaneously measure their spectrum. This is known as hyperspectral microscopy. In this tutorial, we describe and carefully explain how to achieve this goal with an optical microscope equipped with a dark-field objective and an optical spectrometer. The images and the scattering spectra of spherical gold nanoparticles with diameters of 90, 70, 50, and 25 nm are recorded. We compare them with the scattering spectra predicted with the Mie formula (LSPR peaks measured at 553, 541, 535, and 534 nm, respectively). The optical images are limited by the diffraction, and this is discussed in the framework of the Abbe equation. We also describe a strategy to easily correlate the optical images with atomic force microscope images of the samples. This allows us to precisely relate the morphology of the nanoparticles with their optical images, their color, and their optical spectrum. The case of non-spherical nanostructures, namely, dimers of nanoparticles, is also discussed. This approach allows a relatively low-cost setup and efficient characterization method that will be helpful for teachers who want to introduce their students to the wide topics of plasmonics. This will also be useful for labs seeking an affordable method to investigate the plasmonic properties of single nanostructures.

Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the -means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised -means algorithm. The unsupervised -means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within residual error margin. Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.

Learning and Predicting Photonic Responses of Plasmonic Nanoparticle Assemblies via Dual Variational Autoencoders.

The application of machine learning is demonstrated for rapid and accurate extraction of plasmonic particles cluster geometries from hyperspectral image data via a dual variational autoencoder (dual-VAE). In this approach, the information is shared between the latent spaces of two VAEs acting on the particle shape data and spectral data, respectively, but enforcing a common encoding on the shape-spectra pairs. It is shown that this approach can establish the relationship between the geometric characteristics of nanoparticles and their far-field photonic responses, demonstrating that hyperspectral darkfield microscopy can be used to accurately predict the geometry (number of particles, arrangement) of a multiparticle assemblies below the diffraction limit in an automated fashion with high fidelity (for monomers (0.96), dimers (0.86), and trimers (0.58). This approach of building structure-property relationships via shared encoding is universal and should have applications to a broader range of materials science and physics problems in imaging of both molecular and nanomaterial systems.

Probing nanoplastics derived from polypropylene face masks with hyperspectral dark-field microscopy

The high worldwide consumption of cheap plastic goods has already resulted in a serious environmental plastic pollution, exacerbated by piling of disposed personal protective equipment because of the recent outbreak of COVID-19. The aim of this study was to assess the feasibility of dark-field hyperspectral microscopy in the 400–1000 wavelength range for detection of nanoplastics derived from weathered polypropylene masks. A surgical mask was separated to layers and exposed to UV radiation (254 nm) for 192 h. Oxidative degradation of the polypropylene was evidenced by ATR FT-IR analysis. UV treatment for 192 h resulted in generation of differently shaped micro- and nano-sized particles, visualized by dark-field microscopy. The presence of nanoparticles was confirmed by AFM studies. The hyperspectral profiles (400–1000 nm) were collected after every 48 h of the UV treatment. The distinct hyperspectral features faded after prolonged UV exposure, but the assignment of some particles to either blue or white layers of mask could still be made based on spectral characteristics.

Hyperspectral dark-field microscopy for pathogen detection based on spectral angle mapping

Early screening for food pathogens, specifically Escherichia coli O157:H7 in ready-to-eat food remains a central challenge, hyperspectral imaging (HSI) has the potential to acquire both image and spectral information of the target and is simple enough to serve as an effective auxiliary technology in food monitoring. Herein, a visual method was developed based on spectral angle mapping (SAM) and dark-field microscopy for rapid detection and quantification of Escherichia coli. First, immunomagnetic nanoparticle (MNP-probe) and gold nanoparticle (AuNP-probe) probes were prepared to form a sandwich complex for efficient concentration of the target bacteria. The complexes were then dropped directly on a glass slide for colony counting with hyperspectral dark field microscopy. The AuNP-probe bound to the complex was counted and SAM was used to evaluate the spectra and HSI image data to distinguish the target pixel from the background. The linear detection range of the method was estimated to be in the range between 6.1 × 101 and 6.1 × 106 CFU/mL in the milk sample, and the detection limit (LOD) of this method was estimated to be ~61 CFU/mL. The average recovery of organisms in the milk sample was 106.0%, indicating that this method can be utilized to detect Escherichia coli in food samples and serve as an effective method to monitor pathogens in a complex sample.

Spatially Resolved Spectroscopic Characterization of Nanostructured Films by Hyperspectral Dark-Field Microscopy.

Nanostructured films have been widely used for preparing various advanced thin-film devices because of their unique electrical, optical, and plasmonic characteristics associated with the nano-size effect. In situ, nondestructive and high-resolution characterization of nanostructured films is essential for optimizing thin-film device performance. In this work, such thin-film characterization was achieved using a hyperspectral dark-field microscope (HSDFM) that was constructed in our laboratory by integrating a hyperspectral imager with a commercial microscope. The HSDFM allows for high-resolution (Δλ = 0.4 nm) spectral analysis of nanostructured samples in the visible-near-infrared region with a spatial resolution as high as 45 nm × 45 nm (corresponding to a single pixel). Four typical samples were investigated with the HSDFM, including the gold nanoplate array, the self-assembled gold nanoparticle (GNP) sub-monolayer, the sol-gel nanoporous titanium dioxide (TiO2) film, and the layer-stacked molybdenum disulfide (MoS2) sheet. According to the experimental results, the plasmon resonance scattering bands for nanoplate clusters are identical with those for individual gold nanoplates, indicating that the gap between adjacent nanoplates is too large to allow plasmonic coupling between them. A different case was observed with the self-assembled GNP sub-monolayer in which the aggregated clusters with the internal plasmonic interaction show a considerable red-shift of the plasmon resonance band relative to the isolated single GNP. In addition, the protein adsorption on the nanoporous TiO2 film was observed to be inhomogeneous on the microscale, and the stepped boundaries of the MoS2 sheet were clearly observed. A quasi-linear dependence of the single-pixel light intensity on the step height was obtained by combining the HSDFM with atomic force microscopy. The minimum thickness detectable by the present HSDFM is 6.5 nm, corresponding to the 10-layer MoS2 film. The work demonstrated the outstanding applicability of the HSDFM for nanostructured film characterization.

Transcorneal delivery of topically applied silver nanoparticles does not delay epithelial wound healing

Silver nanoparticles (AgNPs) are a common antimicrobial additive for a variety of applications, including wound care. However, AgNPs often undergo dissolution resulting in release of silver ions, with subsequent toxicity to mammalian cells. The cornea is a primary exposure site to topically administered AgNPs in and around the eye but their impact on corneal wound healing is understudied. Thus, the purpose of this study was to determine in vitro toxicity of AgNPs on corneal epithelial cells and fibroblasts as well as their effects on corneal epithelial wound healing utilizing an in vivo rabbit model. Non-coated 20 nm sized AgNP (AgNP-20) as well as 1% and 10% silver silica NPs (AgSiO2NPs) were tested at concentrations ranging from 0.05–250 μg/mL. Immortalized human corneal epithelial (hTCEpi) cells and primary rabbit corneal fibroblasts (RCFs) were incubated for 24 h with AgNPs and cell viability was tested. Additionally, a round wound healing assay was performed to determine hTCEpi cell migration. Quantitative real-time PCR and western blot analysis was performed to determine α-smooth muscle actin (α-SMA, a myofibroblast marker) mRNA and protein expression, respectively, in RCFs treated with 50 μg/mL of AgNPs. Corneal epithelial wound healing was evaluated with 1%-AgSiO2NPs (10 and 250 μg/mL) using an in vivo rabbit model. Rabbits were subsequently euthanized, and histologic sections of the enucleated globes were used to determine corneal penetration of 1%-AgSiO2NPs with autometallography and hyperspectral darkfield microscopy. Cell viability of both the hTCEpi cells and fibroblasts was significantly decreased by the three AgNPs in a dose dependent manner. Migration of hTCEpi cells was significantly inhibited by the three AgNPs. Alpha-SMA mRNA expression was significantly inhibited with three AgNPs, but only the 1%-AgSiO2NPs inhibited protein expression of α-SMA. In vivo epithelial wound closure did not significantly differ between groups treated with 10 or 250 μg/mL of 1%-AgSiO2NPs or vehicle control. The 1%-AgSiO2NPs penetrated throughout all corneal layers and into the anterior chamber in all treated eyes with no histopathological changes observed. In conclusion, the 1%-AgSiO2NPs are safe and have potential therapeutic applications through its efficacy of the corneal penetration and reduced scar formation during corneal wound healing.

The electrochemical dissolution of single silver nanoparticles enlightened by hyperspectral dark-field microscopy

Nowadays, silver nanoparticles are extensively employed in several branches of industry and medicine. Hence, those particles are amongst the most studied class of nanomaterials, yet their reactivity and in particular their reactivity in biological systems is still poorly understood. This discrepancy leads to a huge demand for further insights into the reaction dynamics of electrochemical reactions. For this purpose, coupled opto- and spectro-electrochemical dark-field microscopy is used herein to study the electrochemical oxidation and dissolution process of individual silver nanoparticles in thiocyanate solutions. It is observed, that upon electrochemical oxidation of silver, a silver thiocyanate complex is formed. This is indicated by a change of both the measured plasmon resonance frequency and scattering intensity, simultaneous to the detection of an oxidative current. Subsequently, this silver pseudo-halide is chemically converted to a silver thiocyanate complex with higher solubility in the presence of high thiocyanate concentrations. This follow-up reaction is only detectable thanks to in situ spectroscopy as no current is associated with this chemical conversion but a distinct change in the spectroscopy response of the individual particles is seen. We were thus able to reveal that the total conversion of silver nanoparticles in the presence of thiocyanate is a multi-step process which lasts much longer than the electrochemical response suggests.

Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress

Prior research has demonstrated cells exposed to silver nanoparticles (AgNPs) undergo endoplasmic reticulum (ER) stress leading to cellular apoptosis and toxicity, however, the fundamental mechanism underlying AgNP-induced ER stress is unknown. We hypothesize the biophysical interactions between AgNPs and adsorbed proteins lead to misfolded proteins to elicit an ER stress response. Our investigation examined rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high density lipoprotein (HDL) or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 nm and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not observed for the 100 nm AgNPs. Hyperspectral darkfield microscopy qualitatively confirmed that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the sub-cellular localization of AgNPs. Overall, these results suggest that AgNP size, surface area and BC formation governs the induction of ER stress and alterations in intracellular trafficking.

Probing Interactions between AuNPs/AgNPs and Giant Unilamellar Vesicles (GUVs) Using Hyperspectral Dark-field Microscopy.

Noble metallic nanoparticles (NPs) such as gold and silver nanoparticles (AuNPs and AgNPs) have been shown to exhibit anti-tumor effect in anti-angiogenesis, photothermal and radio therapeutics. On the other hand, cell membranes are critical locales for specific targeting of cancerous cells. Therefore, NP-membrane interactions need be studied at molecular level to help better understand the underlying physicochemical mechanisms for future applications in cancer nanotechnology. Herein, we report our study on the interactions between citrate stabilized colloidal AuNPs/AgNPs (10 nm in size) and giant unilamellar vesicles (GUVs) using hyperspectral dark-field microscopy. GUVs are large model vesicle systems well established for the study of membrane dynamics. GUVs used in this study were prepared with dimyristoyl phosphatidylcholine (DMPC) and doped with cholesterol at various molar concentrations. Both imaging and spectral results support that AuNPs and AgNPs interact very differently with GUVs, i.e., AuNPs tend to integrate in between the lipid bilayer and form a uniform golden-brown crust on vesicles, whereas AgNPs are bejeweled on the vesicle surface as isolated particles or clusters with much varied configurations. The more disruptive capability of AuNPs is hypothesized to be responsible for the formation of golden brown crusts in AuNP-GUV interaction. GUVs of 20 mol% CHOL:DMPC were found to be a most economical concentration for GUVs to achieve the best integrity and the least permeability, consistent with the finding from other phase studies of lipid mixture that the liquid-ordered domains have the largest area fraction of the entire membrane at around 20 mol% of cholesterol.

Photocatalytic Activity of Reduced Graphene Oxide–Gold Nanoparticle Nanomaterials: Interaction with Asphaltene and Conversion of a Model Compound

Asphaltenes are residual materials from the oil industry and are usually converted, after exhaustive distillation, into coke and asphalt. However, conversion of asphaltenes into more valuable raw materials, for instance, by photocatalytic cracking using suitable catalysts, would be a better, more economic option. To explore this idea, we combined the electronic and chemical properties of asphaltene and graphene derivatives with the plasmonic nature of gold nanoparticles. For this purpose, a hybrid material was generated in situ, containing reduced graphene oxide and gold nanoparticles (RGO@AuNP). Evaluation of the interaction between the hybrid material and asphaltenes by hyperspectral dark-field optical microscopy indicated the occurrence of charge transfer between the two species. Using 9-anthraldehyde (9-ATA) as a model compound for asphaltene, photocatalytic experiments performed with RGO@AuNP at room temperature, under visible light irradiation, revealed the formation of cyclic endoperoxides, which u...

High predictive values of RBC membrane-based diagnostics by biophotonics in an integrated approach for Autism Spectrum Disorders

Membranes attract attention in medicine, concerning lipidome composition and fatty acid correlation with neurological diseases. Hyperspectral dark field microscopy (HDFM), a biophotonic imaging using reflectance spectra, provides accurate characterization of healthy adult RBC identifying a library of 8 spectral end-members. Here we report hyperspectral RBC imaging in children affected by Autism Spectrum Disorder (ASD) (n = 21) compared to healthy age-matched subjects (n = 20), investigating if statistically significant differences in their HDFM spectra exist, that can comprehensively map a membrane impairment involved in disease. A significant difference concerning one end-member (spectrum 4) was found (P value = 0.0021). A thorough statistical treatment evidenced: i) diagnostic performance by the receiving operators curve (ROC) analysis, with cut-offs and very high predictive values (P value = 0.0008) of spectrum 4 for identifying disease; ii) significant correlations of spectrum 4 with clinical parameters and with the RBC membrane deficit of the omega-3 docosahexaenoic acid (DHA) in ASD patients; iii) by principal component analysis, very high affinity values of spectrum 4 to the factor that combines behavioural parameters and the variable “cc” discriminating cases and controls. These results foresee the use of biophotonic methodologies in ASD diagnostic panels combining with molecular elements for a correct neuronal growth.

High-resolution contrast-enhanced optical coherence tomography in mice retinae.

Optical coherence tomography (OCT) is a noninvasive interferometric imaging modality providing anatomical information at depths of millimeters and a resolution of micrometers. Conventional OCT images limit our knowledge to anatomical structures alone, without any contrast enhancement. Therefore, here we have, for the first time, optimized an OCT-based contrast-enhanced imaging system for imaging single cells and blood vessels in vivo inside the living mouse retina at subnanomolar sensitivity. We used bioconjugated gold nanorods (GNRs) as exogenous OCT contrast agents. Specifically, we used anti-mouse CD45 coated GNRs to label mouse leukocytes and mPEG-coated GNRs to determine sensitivity of GNR detection in vivo inside mice retinae. We corroborated OCT observations with hyperspectral dark-field microscopy of formalin-fixed histological sections. Our results show that mouse leukocytes that otherwise do not produce OCT contrast can be labeled with GNRs leading to significant OCT intensity equivalent to a 0.5 nM GNR solution. Furthermore, GNRs injected intravenously can be detected inside retinal blood vessels at a sensitivity of ∼0.5 nM, and GNR-labeled cells injected intravenously can be detected inside retinal capillaries by enhanced OCT contrast. We envision the unprecedented resolution and sensitivity of functionalized GNRs coupled with OCT to be adopted for longitudinal studies of retinal disorders.

Building up spectral libraries for mapping erythrocytes by hyperspectral dark field microscopy

BACKGROUND: Red blood cells (RBC) are obtained by non-invasive methods and widely used for diagnostic tests of health status. Hyperspectral Dark Field Microscopy (HDFM) is a promising technique for nanoscale bio imaging and spectral analysis without additional sample preparation. OBJECTIVE: Develop a protocol for human RBC characterization by HDFM, checking the feasibility of a reference spectral library that can image and afford a new comprehensive descriptor of RBC status. METHOD: A step-by-step protocol for HDFM measurement of human RBC was for the first time established using 5 µl of EDTA-treated whole blood from healthy adults (n = 30). Hyperspectral characteristics of solutions/suspensions at biological concentrations of phospholipids, hemoglobin, spectrin, cholesterol and protoporphyrin IX, as the most relevant RBC components, were also determined. RESULTS: A library made of 8 end-member spectra and classification of their spectral distribution carried out by Single Angle Mapper (SAM) were determined, furnishing a comprehensive mapping and descriptor of healthy human RBC. The spectra of single components allowed some of the RBC spectral bands to be attributed. CONCLUSIONS: This work reports for the first time the hyperspectral optical imaging of the human RBC by a library made of 8 scattering spectra, whose spectral signatures are compared with those of the main RBC molecular components. The percent distribution of the spectral end-members was also achieved, thus giving for the first time the HDFM mapping of human healthy RBCs. The protocol developed herein allows the clinical potential of hyperspectral imaging to be developed for the use of RBC mapping in health and disease.

Optical Clearing Delivers Ultrasensitive Hyperspectral Dark-Field Imaging for Single-Cell Evaluation.

A single-cell optical clearing methodology is developed and demonstrated in hyperspectral dark-field microscopy (HSDFM) and imaging of plasmonic nanoprobes. Our strategy relies on a combination of delipidation and refractive index (RI) matching with highly biocompatible and affordable agents. Before applying the RI-matching solution, the delipidation step by using a mild solvent effectively eliminates those high-density, lipid-enriched granular structures which emit strong scattering. Upon treatment, the background scattering from cellular organelles could be repressed to a negligible level while the scattering signals from plasmonic nanomaterials increase, leading to a significant improvement of the signal-to-noise ratio (SNR). With this method established, the versatility and applicability of HSDFM are greatly enhanced. In our demonstration, quantitative mapping of the dimerization-activated receptor kinase HER2 is achieved in a single cancer cell by a nonfluorescent approach. High-resolution imaging for oncogenic mRNAs, namely ER, PR, and HER2, is performed with single labeling. More importantly, in situ multiplex detection of mRNA and protein is made possible by HSDFM since it overcomes the difficulties of complex staining and signal imbalance suffered by the conventional optical imaging. Last, we show that with optical clearing, characterization of intracellularly grown gold particulates is accomplished at an unprecedented spatiotemporal resolution. Taken together, the uniqueness of optical clearing and HSDFM is expected to open ample avenues for single-cell studies and biomedical engineering.

Scattering based hyperspectral imaging of plasmonic nanoplate clusters towards biomedical applications.

A new optical scattering contrast-agent based on polymer-nanoparticle encapsulated silver nanoplates (PESNs) is presented. Silver nanoplates were chosen due to the flexibility of tuning their plasmon frequencies. The polymer coating preserves their physical and optical properties and confers other advantages such as controlled contrast agent delivery. Finite difference time domain (FDTD) simulations model the interaction of light with the nanoplates in different orientations in the cluster. Hyperspectral dark field microscopy (HYDFM) observes the scattering spectra of the PESNs. An unsupervised sequential maximum angle convex cone (SMACC) image analysis resolves spectral endmembers corresponding to different stacking orientations of the nanoplates. The orientation-dependent endmembers qualitatively agree with the FDTD results. For contrast enhancement, the uptake and spatial distribution of PESNs are demonstrated by an HYDFM study of single melanoma cells to result in an enhanced contrast of up to 400%. A supervised spatial mapping of the endmembers obtained by the unsupervised SMACC algorithm reveals spatial distributions of PESNs with various clustering orientations of encapsulated nanoplates. Our study demonstrates tunability in plasmonics properties in clustered metal nanoparticles and its utility for the development of scatter-based imaging contrast agents for a broad range of applications, including studies of single cells and other biomedical systems.

Hyperspectral dark-field microscopy of gold nanodisks

The light scattering properties of hexagonal and triangular gold nanodisks were investigated by means of Cytoviva hyperspectral dark-field microscopy, exploring the huge enhancement of the scattered waves associated with the surface plasmon resonance (SPR) effect. Thanks to the high resolution capability of the dark-field microscope, the SPR effect turned it possible to probe the individual nanoparticles directly from their hyperspectral images, extrapolating the classical optical resolution limit, and providing their corresponding extinction spectra. Blue spectral shifts involving the in-plane dipolar modes were observed for the hexagonal gold nanodisks in relation to the triangular ones, allowing their spectroscopic differentiation in the dark-field images.

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44(+) human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub-cellular activity with the use of NPs as spectral sensors.

How relevant can the SERS effect in isolated nanoparticles be?

Gold nanoparticles electrostatically stabilized with negatively charged ruthenium complexes were individually monitored based on their characteristic plasmon bands, by means of hyperspectral dark field microscopy. Very strong SERS enhancements were observed for the isolated gold nanoparticles at exciting wavelengths in resonance with the surface plasmon band, using confocal Raman microscopy, revealing a contrasting behaviour between the agglomerated and non-agglomerated systems. The results highlighted the relevant role played by the surface enhanced resonance Raman mechanisms (SERRS) in isolated nanoparticles, more than compensating for the lack of local hot spots, in relation to the agglomerated systems.

Hyperspectral darkfield microscopy of single hollow gold nanoparticles for biomedical applications

Hyperspectral microscopy is a versatile method for simultaneous spatial and spectroscopic characterization of nonfluorescent samples. Here we present a hyperspectral darkfield imaging system for spectral imaging of single nanoparticles over an area of 150 × 150 μm(2) and at illumination intensities compatible with live cell imaging. The capabilities of the system are demonstrated using correlated transmission electron microscopy and single-particle optical studies of colloidal hollow gold nanoparticles. The potential of the system for characterizing the interactions between nanoparticles and cells has also been demonstrated. In this case, the spectral information proves a useful improvement to standard darkfield imaging as it enables differentiation between light scattered from nanoparticles and light scattered from other sources in the cellular environment. The combination of low illumination power and fast integration times makes the system highly suitable for nanoparticle tracking and spectroscopy in live-cell experiments.