Characterising the chemical and physical properties of phase-change nanodroplets.

Phase-change nanodroplets have attracted increasing interest in recent years as ultrasound theranostic nanoparticles. They are smaller compared to microbubbles and they may distribute better in tissues (e.g. in tumours). They are composed of a stabilising shell and a perfluorocarbon core. Nanodroplets can vaporise into echogenic microbubbles forming cavitation nuclei when exposed to ultrasound. Their perfluorocarbon core phase-change is responsible for the acoustic droplet vaporisation. However, methods to quantify the perfluorocarbon core in nanodroplets are lacking. This is an important feature that can help explain nanodroplet phase change characteristics. In this study, we fabricated nanodroplets using lipids shell and perfluorocarbons. To assess the amount of perfluorocarbon in the core we used two methods, 19F NMR and FTIR. To assess the cavitation after vaporisation we used an ultrasound transducer (1.1 MHz) and a high-speed camera. The 19F NMR based method showed that the fluorine signal correlated accurately with the perfluorocarbon concentration. Using this correlation, we were able to quantify the perfluorocarbon core of nanodroplets. This method was used to assess the content of the perfluorocarbon of the nanodroplets in solutions over time. It was found that perfluoropentane nanodroplets lost their content faster and at higher ratio compared to perfluorohexane nanodroplets. The high-speed imaging indicates that the nanodroplets generate cavitation comparable to that from commercial contrast agent microbubbles. Nanodroplet characterisation should include perfluorocarbon concentration assessment as critical information for their development.

Label-free study of intracellular glycogen level in metformin and resveratrol-treated insulin-resistant HepG2 by live-cell FTIR spectroscopy.

Conventional in vitro study often involves the destruction of the cells followed by purification and dilution steps before applying enzymatic assay or metabolomic analysis. It is a costly and laborious process, and it cannot monitor changes as a function of time. Recently, we have developed a new label-free live-cell FTIR approach that can directly measure biochemical compositional changes within living cells in situ and the spectral changes are shown to be highly specific to the drug applied. In this work, we have demonstrated for the first time the effect of two anti-diabetic drugs, metformin and Resveratrol, on insulin-resistant liver cells (HepG2). Using live-cell FTIR with principal component analysis, we have shown the differences in the biochemical profiles between normal and insulin-resistant cells (p < 0.05), the lack of response/difference from the insulin-resistant cell to insulin (p > 0.05) and the restoration of the biochemical profile and sensitivity to insulin from the insulin-resistant cells after the drug treatment (p < 0.05). Particularly, a rise in the glycogen level, marked by three distinctive peaks at 1150, 1080 and 1020 cm-1, within the living cells after the anti-diabetic drug treatments is observed. The live-cell FTIR results are confirmed by a parallel gold-standard biochemical assay, demonstrating the restoration of insulin sensitivity of the insulin-resistance cells. Live-cell FTIR can be a complementary tool for drug efficacy screening, especially for insulin sensitizers.

Amino acid hydrotropes to increase the solubility of indomethacin and carbamazepine in aqueous solution.

A number of amino acids (AA) has been investigated as promising hydrotropes to improve the solubility of biopharmaceutics classification system (BCS) class II drugs carbamazepine (CBZ) and indomethacin (IND) via specific complexations in aqueous solution. The aim of this work is to understand the molecular basis of these hydrotropic interactions by investigating the two model drugs combined with 12 amino acids including phenylalanine, tryptophan, isoleucine, proline, valine, glycine, serine, threonine, arginine, lysine, histidine and aspartic acid in water at 25 °C, 30 °C and 45 °C. The amino acids were chosen based on their different side chains (neutral aromatic, aliphatic, polar charged or uncharged) to investigate their hydrotropic performance. A linear solubility curve was observed between indomethacin and mono-neutral hydrophobic amino acids (phenylalanine, tryptophan, isoleucine, proline and valine) well beyond 1:1 molar ratio indicating the interaction is predominantly non-ionic between the drug and the hydrotropes. Interestingly, the aqueous solubility of carbamazepine (a neutral compound) was enhanced by neutral, charged basic or acidic amino acids, confirming the presence of hydrophobic interactions that involve H-bonds, H/π and π/π stacking and the results were confirmed by UV-Vis spectroscopy. A combination of multiple neutral amino acids showed additive hydrotropic effect in indomethacin solubility with up to 7-folds increases. This study demonstrates for the first time the potential of amino acids as hydrotropes to improve aqueous solubility of poorly water-soluble drugs, which is important for pharmaceutical development.

In vitro Fourier transform infrared spectroscopic study of the effect of glycerol on the uptake of beclomethasone dipropionate in living respiratory cells.

The quantification of drug in living cells is of increasing interest in pharmaceutical research because of its importance in understanding drug efficacy and toxicity. Label-free in situ measurement methods are advantageous for their ability to obtain chemical and time profiles without the need of labelling or extraction steps. We have previously shown that Fourier transform infrared (FTIR) spectroscopy has the potential to quantify drug in situ within living cells at micromolar level when a simple solution of drug was added to the medium. The purpose of this study was to demonstrate that the approach can evaluate more complex systems such as the effect of membrane modification by a formulation on drug uptakes. The inhaled corticosteroid, beclomethasone dipropionate (BDP), in Calu-3 respiratory epithelial cells in the absence and presence of glycerol, an excipient in some inhaled medicines was used as the model system. The FTIR method was first validated for limit of detection (LOD) and quantification (LOQ) according to published guidelines and the LOQ was found to be âˆ¼ 20 µM, good enough to quantify BDP in the living cell. The uptake of BDP by living Calu-3 cells was found to be reduced in the presence of glycerol as expected due to the stiffening of the cell membrane by the presence of glycerol in the formulation. This study demonstrates the valuable analytical capability of live-cell FTIR to study the effect of formulation on drug transport in lungs and to evaluate drug availability to intracellular targets. We conclude that FTIR has potential to contribute widely at the frontier of live-cell studies.

Live-cell ATR-FTIR spectroscopy as a novel bioanalytical tool for cell glucose metabolism research.

Current novel drug developments for the treatment of diabetes require multiple bioanalytical assays to interrogate the cellular metabolism, which are costly, laborious and time-consuming. Fourier-transform infrared (FTIR) spectroscopy is a nondestructive, label-free, sensitive and low-cost technique that is recently found to be suitable for studying living cells. The aim of this study is to demonstrate that live-cell FTIR can be applied to study the differences in glucose metabolism in cells in normal culturing medium and cells treated in high glucose (a diabetes model) in order to highlight the potential of the technique in diabetes research. Live HepG2 cells were treated in normal glucose (3.8 mM; control) or high glucose (25 mM) medium and were measured directly using the FTIR approach. Principal component analysis was used to highlight any possible correlated changes 24, 48 and 72 h after treatments. FTIR spectra of live cell treated in normal and high glucose medium have shown significant differences (p < 0.05) for all treatment time. The control cells have seen an increased in the absorbance at 1088, 1240 and 1400 cm-1, which are associated with phosphate stretching mode vibrations from phosphorylated proteins and DNA back bone; and symmetric stretching mode vibration of COO- from fatty acids, amino acids, lipids and carbohydrate metabolites. However, the high glucose treated cells have shown a different changes in the 1000-1200 cm-1 region, which is linked to the glycogen and ATP:ADP ratio. In conclusion, live-cell FTIR can be a low-cost method for the studies of metabolic changes in cells.

Tracking glycosylation in live cells using FTIR spectroscopy.

This is the first demonstration of the study of glycan protein turnover in living cells by FTIR with commercially available tetraacetylated N-Azidoacetyl-D-Mannosamine (Ac4ManNAz) label. The FTIR analysis has shown to be able to monitor the metabolism of glycans in living cells in real time. The method is simple, quantitative and requires equipment that are available in many laboratories. It can be used in a wide range of applications such as the study of glycosylation and cell-signalling.

Towards identifying the mode of action of drugs using live-cell FTIR spectroscopy.

Fourier transform infrared spectroscopy (FTIR) has been shown to be a promising tool for identifying the mode of action of drugs. However, most previous studies have focused on the analysis of fixed or dried cells. The measurement of living cells has the advantage of obtaining time series data, and the in situ approach eliminates the need for fixing or drying the cells. In this study, the potential of live-cell FTIR method for the identification of the mode of action of drugs was demonstrated. Four different drugs were tested, with two of the drugs having the same mode of action (tamoxifen and toremifene) and the other two having different modes of action (imatinib and doxorubicin). Live cells were treated in the four drugs at and below the IC50 level (i.e. the concentration of drug required to inhibit the growth of cells by 50%), and the changes to their spectra after the addition of drugs were monitored over a 24-hour period. Principal component analysis (PCA) of the spectral data shows that drugs with different modes of action are well-separated, while the drugs with the same mode of action are grouped together. The results also show that at IC50, the separation appears to be the clearest at 2 hours for imatinib and tamoxifen/toremifene and 6 hours for doxorubicin. However, at 50% of the IC50 drug concentration, the separation appears to be the best at longer incubation time, i.e. 24 hours, for all four drugs. In conclusion, live-cell FTIR has shown to be able to distinguish and group spectral signatures of cells treated with drugs of known modes of action after a relatively short time of exposure. Further collection of live-cell data would enable an algorithm to be developed for the prediction of the modes of action of novel drugs, which can help in the preclinical drug screening process.

Subcellular mapping of living cells via synchrotron microFTIR and ZnS hemispheres.

FTIR imaging is a label-free, non-destructive method valuably exploited in the study of the biological process in living cells. However, the long wavelength/low spatial resolution and the strong absorbance of water are still key constrains in the application of IR microscopy ex vivo. In this work, a new retrofit approach based on the use of ZnS hemispheres is introduced to significantly improve the spatial resolution on live cell FTIR imaging. By means of two high refractive index domes sandwiching the sample, a lateral resolution close to 2.2 µm at 6 µm wavelength has been achieved, i.e. below the theoretical diffraction limit in air and more than twice the improvement (to ~λ/2.7) from our previous attempt using CaF2 lenses. The ZnS domes also allowed an extended spectral range to 950 cm-1, in contrast to the cut-off at 1050 cm-1 using CaF2. In combination with synchrotron radiation source, microFTIR provides an improved signal-to-noise ratio through the circa 12 µm thin layer of medium, thus allowing detailed distribution of lipids, protein and nucleic acid in the surround of the nucleus of single living cells. Endoplasmic reticula were clearly shown based on the lipid ν(CH) and ν(C=O) bands, while the DNA was imaged based on the ν(PO2-) band highlighting the nucleus region. This work has also included a demonstration of drug (doxorubicin) in cell measurement to highlight the potential of this approach. Graphical abstract.

A combined FTIR and DSC study on the bilayer-stabilising effect of electrostatic interactions in ion paired lipids.

Investigating lipid ion pair formation is important for understanding the mechanisms of lipid-mediated drug resistance in bacteria. In this study we have used the charged amphiphiles dipalmitoylphosphatidylglycerol (DPPG) and dihexadecyldimethylammonium bromide (DHDAB), as a model to evaluate the formation of ion pairs by a combined Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analysis. FTIR was employed to study the environment of the DPPC headgroup phosphate and lipid/surfactant alkane chains, in vesicles formed by the two amphiphiles mixed in various molar ratios. An increase of the absorbance ratio of 1221-1201 cm-1 in the asymmetric phosphate stretching mode was found to follow a sigmoidal relationship with the proportion of DHDAB, increasing to a plateau above a DPPG/DHDAB 1:1 molar ratio of, providing evidence that the PG headgroup phosphate is involved in ion pairing. A consistent red shift was measured for the position of the symmetric CH2 stretch band for the lipid/surfactant 1:1 molar ratio mixture, which is indicative of an increased ordering of the hydrophobic chains. The DSC experiments yielded information about the thermotropic and the mixing behaviour of the lipid/surfactant systems. DPPG and DHDAB seem to form an ion pair with cluster compound characteristics at the equimolar ratio. Most interestingly, the DPPG/DHDAB 2:1 molar ratio mixture is characterized by strong intermolecular interactions, which result in a pronounced stabilization of the gel phase, possibly through the formation of a closely-associated ion triplet configuration in which the charges are delocalised across the headgroups.

In-situ freeze-drying - forming amorphous solids directly within capsules: An investigation of dissolution enhancement for a poorly soluble drug.

Conversion into the amorphous form enhances the dissolution of poorly soluble drugs, however the barrier to market for medicines containing an amorphous drug is poor stability. The aim was to produce the amorphous form of a drug within a capsule, without thermal or mechanical stress during manufacture. To facilitate this aim, the mechanism for drug-polymer interaction was explored. Nifedipine and polyvinylpyrrolidone were dissolved in tert-butanol at different drug/polymer ratios. These solutions were dispensed into gelatin capsules and freeze-dried. Differential scanning calorimetry (DSC) & novel FT-IR analysis based on peak symmetry measurements confirmed the absence of crystallinity when polyvinylpyrrolidone exceeded 50%w/w. Capsules containing 10 mg of nifedipine were amorphous and stable for over 3 months at ≈40 °C. Evidence of hydrogen bonding between the N-H group of nifedipine and the C=O group of PVP was observed and this interaction inhibited nifedipine crystallisation. PVP's high affinity for water and the nifedipine-polymer interaction lead to a significant dissolution rate enhancement. The freeze-dried capsule, 10%w/w nifedipine/PVP, had the highest dissolution rate constant of 0.37 ± 0.05 min-1, and the lowest time to achieve 50% dissolution or t1/2 of 1.88 ± 0.05 min. This formulation reached 80% dissolved in less than 6 min whereas the equivalent marketed liquid filled nifedipine capsule took 3 times longer to reach 80% dissolution.

In situ Fourier transform infrared analysis of live cells' response to doxorubicin.

The study of the response of cancer cells to chemotherapy drugs is of high importance due to the specificity of some drugs to certain types of cancer and the resistance of some specific cancer types to chemotherapy drugs. Our aim was to develop and apply the label-free and non-destructive Fourier transform infrared (FTIR) method to determine the sensitivity of three different cancer cell-lines to a common anti-cancer drug doxorubicin at different concentrations and to demonstrate that information about the mechanism of resistance to the chemotherapy drug can be extracted from spectral data. HeLa, PC3, and Caco-2 cells were seeded and grown on an attenuated total reflection (ATR) crystal, doxorubicin was applied at the clinically significant concentration of 0.1-20 µM, and spectra of the cells were collected hourly over 20 h. Analysis of the amide bands was correlated with cell viability, which had been cross validated with MTT assays, allowing to determine that the three cell lines had significantly different resistance to doxorubicin. The difference spectra and principal component analysis (PCA) highlighted the subtle chemical changes in the living cells under treatment. Spectral regions assigned to nucleic acids (mainly 1085 cm(-1)) and carbohydrates (mainly 1024 cm(-1)) showed changes that could be related to the mode of action of the drug and the mechanism of resistance of the cell lines to doxorubicin. This is a cost-effective method that does not require bioassay reagents but allows label-free, non-destructive and in situ analysis of chemical changes in live cells, using standard FTIR equipment adapted to ATR measurements.

A central neural pathway controlling odor tracking in Drosophila.

Chemotaxis is important for the survival of most animals. How the brain translates sensory input into motor output beyond higher olfactory processing centers is largely unknown. We describe a group of excitatory neurons, termed Odd neurons, which are important for Drosophila larval chemotaxis. Odd neurons receive synaptic input from projection neurons in the calyx of the mushroom body and project axons to the central brain. Functional imaging shows that some of the Odd neurons respond to odor. Larvae in which Odd neurons are silenced are less efficient at odor tracking than controls and sample the odor space more frequently. Larvae in which the excitability of Odd neurons is increased are better at odor intensity discrimination and odor tracking. Thus, the Odd neurons represent a distinct pathway that regulates the sensitivity of the olfactory system to odor concentrations, demonstrating that efficient chemotaxis depends on processing of odor strength downstream of higher olfactory centers.

Label-free in situ quantification of drug in living cells at micromolar levels using infrared spectroscopy.

Quantifying the rate and the amount of drug entering live cells is an essential part of the medicine development process. Infrared spectroscopy is a label-free, chemically selective tool for analyzing the composition of live cells in culture that has the potential to quantify, in situ, the amount of drug entering living cells in a nondestructive manner, although its sensitivity is currently limited. This paper is the first to demonstrate in situ quantification of the cancer drug, fluorouracil, in live cells at a therapeutically relevant concentration using Fourier transform infrared spectroscopy. To achieve the required improvement in detection and quantitation limits of the IR measurement, two strategies were exploited. First, a sampling method called multibounce attenuated total reflection was used to optimize the signal while second, a long pass filter in combination with a mercury cadmium telluride detector was used to reduce the instrument noise. Using these novel adaptations, it was possible to quantify 20 µM of fluorouracil in cell culture medium using a standard FTIR instrument, while it was possible to quantify and measure the flux of fluorouracil in situ in living cells treated with an 80 µM drug.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

ATR-FTIR spectroscopic imaging: recent advances and applications to biological systems.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging is a highly versatile, label free and non-destructive chemical imaging method which can be applied to study a wide range of samples and systems. This review summarises some of the recent advances and applications of this imaging method in the area of biomedical studies, including examples of section of aorta, skin tissue and live cells. Two of the major advantages of measuring in ATR mode are the opportunity to measure samples that absorb strongly in the IR spectrum, such as aqueous systems, without significant sample preparation and the ability to increase the spatial resolution of the measured image. The implications of these advantages as well as some limitations of this imaging approach are discussed and a brief outlook at some of the possible future developments in this area is provided.

Correcting the effect of refraction and dispersion of light in FT-IR spectroscopic imaging in transmission through thick infrared windows.

Transmission mode is one of the most common sampling methods for FT-IR spectroscopic imaging because the spectra obtained generally have a reasonable signal-to-noise ratio. However, dispersion and refraction of infrared light occurs when samples are sandwiched between infrared windows or placed underneath a layer of liquid. Dispersion and refraction cause infrared light to focus with different focal lengths depending on the wavelength (wavenumber) of the light. As a result, images obtained are in focus only at a particular wavenumber while they are defocused at other wavenumber values. In this work, a solution to correct this spread of focus by means of adding a lens on top of the infrared transparent window, such that a pseudo hemisphere is formed, has been investigated. Through this lens (or pseudo hemisphere), refraction of light is removed and the light across the spectral range has the same focal depth. Furthermore, the lens acts as a solid immersion objective and an increase of both magnification and spatial resolution (by 1.4 times) is demonstrated. The spatial resolution was investigated using an USAF resolution target, showing that the Rayleigh criterion can be achieved, as well as a sample with a sharp polymer interface to indicate the spatial resolution that can be expected in real samples. The reported approach was used to obtain chemical images of cross sections of cancer tissue and hair samples sandwiched between infrared windows showing the versatility and applicability of the method. In addition to the improved spatial resolution, the results reported herein also demonstrate that the lens can reduce the effect of scattering near the edges of tissue samples. The advantages of the presented approach, obtaining FT-IR spectroscopic images in transmission mode with the same focus across all wavenumber values and simultaneous improvement in spatial resolution, will have wide implications ranging from studies of live cells to sorption of drugs into tissues.

FT-IR spectroscopic imaging of reactions in multiphase flow in microfluidic channels.

Rapid, in situ, and label-free chemical analysis in microfluidic devices is highly desirable. FT-IR spectroscopic imaging has previously been shown to be a powerful tool to visualize the distribution of different chemicals in flows in a microfluidic device at near video rate imaging speed without tracers or dyes. This paper demonstrates the possibility of using this imaging technology to capture the chemical information of all reactants and products at different points in time and space in a two-phase system. Differences in the rates of chemical reactions in laminar flow and segmented flow systems are also compared. Neutralization of benzoic acid in decanol with disodium phosphate in water has been used as the model reaction. Quantitative information, such as concentration profiles of reactant and products, can be extracted from the imaging data. The same feed flow rate was used in both the laminar flow and segmented flow systems. The laminar flow pattern was achieved using a plain wide T-junction, whereas the segmented flow was achieved by introducing a narrowed section and a nozzle at the T-junction. The results show that the reaction rate is limited by diffusion and is much slower with the laminar flow pattern, whereas the reaction is completed more quickly in the segmented flow due to better mixing.

Chemical imaging of protein adsorption and crystallization on a wettability gradient surface.

The use of self-assembled monolayers is an established method to study the effect of surface properties on proteins and other biological materials. The generation of a monolayer with a gradient of chemical properties allows for the study of multiple surface properties simultaneously in a high throughput manner. Typically, in order to detect the presence of proteins or biological material on a surface, the use of additional dyes or tags is required. Here we present a novel method of studying the effect of gradient surface properties on protein adsorption and crystallization in situ through the use of ATR-FTIR spectroscopic imaging, which removes the need for additional labeling. We describe the successful application of this technique to the measurement of the growth of a gradient monolayer of octyltrichlorosilane across the surface of a silicon ATR element. ATR-FTIR imaging was also used to study the adsorption of lysozyme, as a model protein, onto the modified surface. The sensitivity of measurements obtained with a focal plane array (FPA) detector were improved though the use of pixel averaging which allowed small absorption bands to be detected with minimal effect on the spatial resolution along the gradient. Study of the effect of surface hydrophobicity on both adsorption of lysozyme to the element and lysozyme crystallization revealed that more lysozyme adsorbed to the hydrophobic side of the ATR element and more lysozyme crystals formed in the same region. These findings strongly suggest a correlation exists between surface protein adsorption and protein crystallization. This method could be applied to the study of other proteins and whole cells.

Mapping local microstructure and mechanical performance around carbon nanotube grafted silica fibres: methodologies for hierarchical composites.

The introduction of carbon nanotubes (CNTs) modifies bulk polymer properties, depending on intrinsic quality, dispersion, alignment, interfacial chemistry and mechanical properties of the nanofiller. These effects can be exploited to enhance the matrices of conventional microscale fibre-reinforced polymer composites, by using primary reinforcing fibres grafted with CNTs. This paper presents a methodology that combines atomic force microscopy, polarised Raman spectroscopy, and nanoindentation techniques, to study the distribution, alignment and orientation of CNTs in the vicinity of epoxy-embedded micrometre-scale silica fibres, as well as, the resulting local mechanical properties of the matrix. Raman maps of key features in the CNT spectra clearly show the CNT distribution and orientation, including a 'parted' morphology associated with long grafted CNTs. The hardness and indentation modulus of the epoxy matrix were improved locally by 28% and 24%, respectively, due to the reinforcing effects of CNTs. Moreover, a slower stress relaxation was observed in the epoxy region containing CNTs, which may be due to restricted molecular mobility of the matrix. The proposed methodology is likely to be relevant to further studies of nanocomposites and hierarchical composites.

Tip-enhanced Raman mapping with top-illumination AFM.

Tip-enhanced Raman mapping is a powerful, emerging technique that offers rich chemical information and high spatial resolution. Currently, most of the successes in tip-enhanced Raman scattering (TERS) measurements are based on the inverted configuration where tips and laser are approaching the sample from opposite sides. This results in the limitation of measurement for transparent samples only. Several approaches have been developed to obtain tip-enhanced Raman mapping in reflection mode, many of which involve certain customisations of the system. We have demonstrated in this work that it is also possible to obtain TERS nano-images using an upright microscope (top-illumination) with a gold-coated Si atomic force microscope (AFM) cantilever without significant modification to the existing integrated AFM/Raman system. A TERS image of a single-walled carbon nanotube has been achieved with a spatial resolution of ∼ 20-50 nm, demonstrating the potential of this technique for studying non-transparent nanoscale materials.