Analysis of the Chemical Distribution of Self-Assembled Microdomains with the Selective Localization of Amine-Functionalized Graphene Nanoplatelets by Optical Photothermal Infrared Microspectroscopy.

By incorporating 1-(2-aminoethyl)piperazine (AEPIP) into a commercial epoxy blend, a bicontinuous microstructure is produced with the selective localization of amine-functionalized graphene nanoplatelets (A-GNPs). This cured blend underwent self-assembly, and the morphology and topology were observed via spectral imaging techniques. As the selective localization of nanofillers in thermoset blends is rarely achieved, and the mechanism remains largely unknown, the optical photothermal infrared (O-PTIR) spectroscopy technique was employed to identify the compositions of microdomains. The A-GNP tends to be located in the region containing higher concentrations of both secondary amine and secondary alcohol; additionally, the phase morphology was found to be influenced by the amine concentration. With the addition of AEPIP, the size of the graphene domains becomes smaller and secondary phase separation is detected within the graphene domain evidenced by the chemical contrast shown in the high-resolution chemical map. The corresponding chemical mapping clearly shows that this phenomenon was mainly induced by the chemical contrast in related regions. The findings reported here provide new insight into a complicated, self-assembled nanofiller domain formed in a multicomponent epoxy blend, demonstrating the potential of O-PTIR as a powerful and useful approach for assessing the mechanism of selectively locating nanofillers in the phase structure of complex thermoset systems.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line.

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and ß-glycerophosphate (ßG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.

Analysis of Hepatic Fibrosis Characteristics in Cirrhotic Patients with and without Hepatocellular Carcinoma by FTIR Spectral Imaging.

The evolution of cirrhosis is marked by quantitative and qualitative modifications of the fibrosis tissue and an increasing risk of complications such as hepatocellular carcinoma (HCC). Our purpose was to identify by FTIR imaging the spectral characteristics of hepatic fibrosis in cirrhotic patients with and without HCC. FTIR images were collected at projected pixel sizes of 25 and 2.7 µm from paraffinized hepatic tissues of five patients with uncomplicated cirrhosis and five cirrhotic patients with HCC and analyzed by k-means clustering. When compared to the adjacent histological section, the spectral clusters corresponding to hepatic fibrosis and regeneration nodules were easily identified. The fibrosis area estimated by FTIR imaging was correlated to that evaluated by digital image analysis of histological sections and was higher in patients with HCC compared to those without complications. Qualitative differences were also observed when fibrosis areas were specifically targeted at higher resolution. The partition in two clusters of the fibrosis tissue highlighted subtle differences in the spectral characteristics of the two groups of patients. These data show that the quantitative and qualitative changes of fibrosis tissue occurring during the course of cirrhosis are detectable by FTIR imaging, suggesting the possibility of subclassifying cirrhosis into different steps of severity.

Calcification Microstructure Reflects Breast Tissue Microenvironment.

Microcalcifications are important diagnostic indicators of disease in breast tissue. Tissue microenvironments differ in many aspects between normal and cancerous cells, notably extracellular pH and glycolytic respiration. Hydroxyapatite microcalcification microstructure is also found to differ between tissue pathologies, including differential ion substitutions and the presence of additional crystallographic phases. Distinguishing between tissue pathologies at an early stage is essential to improve patient experience and diagnostic accuracy, leading to better disease outcome. This study explores the hypothesis that microenvironment features may become immortalised within calcification crystallite characteristics thus becoming indicators of tissue pathology. In total, 55 breast calcifications incorporating 3 tissue pathologies (benign - B2, ductal carcinoma in-situ - B5a and invasive malignancy - B5b) from archive formalin-fixed paraffin-embedded core needle breast biopsies were analysed using X-ray diffraction. Crystallite size and strain were determined from 548 diffractograms using Williamson-Hall analysis. There was an increased crystallinity of hydroxyapatite with tissue malignancy compared to benign tissue. Coherence length was significantly correlated with pathology grade in all basis crystallographic directions (P < 0.01), with a greater difference between benign and in situ disease compared to in-situ disease and invasive malignancy. Crystallite size and non-uniform strain contributed to peak broadening in all three pathologies. Furthermore, crystallite size and non-uniform strain normal to the basal planes increased significantly with malignancy (P < 0.05). Our findings support the view that tissue microenvironments can influence differing formation mechanisms of hydroxyapatite through acidic precursors, leading to differential substitution of carbonate into the hydroxide and phosphate sites, causing significant changes in crystallite size and non-uniform strain.

Assessment of Compressive Raman versus Hyperspectral Raman for Microcalcification Chemical Imaging.

We experimentally implement a compressive Raman technology (CRT) that incorporates chemometric analysis directly into the spectrometer hardware by means of a digital micromirror device (DMD). The DMD is a programmable optical filter on which optimized binary filters are displayed. The latter are generated with an algorithm based on the Cramer-Rao lower bound. We compared the developed CRT microspectrometer with two conventional state-of-the-art Raman hyperspectral imaging systems on samples mimicking microcalcifications relevant for breast cancer diagnosis. The CRT limit of detection significantly improves, when compared to the CCD based system, and CRT ultimately allows 100× and 10× faster acquisition speeds than the CCD- and EMCCD-based systems, respectively.

Taking a Tailored Approach to Material Design: A Mechanistic Study of the Selective Localization of Phase-Separated Graphene Microdomains.

To achieve multifunctional properties using nanocomposites, selectively locating nanofillers in specific areas by tailoring a mixture of two immiscible polymers has been widely investigated. Forming a phase-separated structure from entirely miscible molecules is rarely reported, and the related mechanisms to govern the formation of assemblies from molecules have not been fully resolved. In this work, a novel method and the underlying mechanism to fabricate self-assembling, bicontinuous, biphasic structures with localized domains made up of amine-functionalized graphene nanoplatelets are presented, involving the tailoring of compositions in a liquid processable multicomponent epoxy blend. Kinetics studies were carried out to investigate the differences in reactivity of various epoxy-hardener pairs. Molecular dynamics simulations and in situ optical photothermal infrared spectroscopy measurements revealed the trajectories of different components during the early stages of polymerization, supporting the migration (phase behavior) of each component during the curing process. Confirmed by the phase structure and the correlated chemical maps down to the submicrometer level, it is believed that the bicontinuous phase separation is driven by the change of the miscibility between various building blocks forming during polymerization, leading to the formation of nanofiller domains. The proposed morphology evolution mechanism is based on combining solubility parameter calculations with kinetics studies, and preliminary experiments are performed to validate the applicability of the mechanism of selectively locating nanofillers in the phase-separated structure. This provides a simple yet sophisticated engineering model and a roadmap to a mechanism for fabricating phase-separated structures with nanofiller domains in nanocomposite films.

Translating microcalcification biomarker information into the laboratory: A preliminary assessment utilizing core biopsies obtained from sites of mammographic calcification.

Rationale and objectives: The potential of breast microcalcification chemistry to provide clinically valuable intelligence is being increasingly studied. However, acquisition of crystallographic details has, to date, been limited to high brightness, synchrotron radiation sources. This study, for the first time, evaluates a laboratory-based system that interrogates histological sections containing microcalcifications. The principal objective was to determine the measurement precision of the laboratory system and assess whether this was sufficient to provide potentially clinical valuable information. Materials and methods: Sections from 5 histological specimens from breast core biopsies obtained to evaluate mammographic calcification were examined using a synchrotron source and a laboratory-based instrument. The samples were chosen to represent a significant proportion of the known breast tissue, mineralogical landscape. Data were subsequently analysed using conventional methods and microcalcification characteristics such as crystallographic phase, chemical deviation from ideal stoichiometry and microstructure were determined. Results: The crystallographic phase of each microcalcification (e.g., hydroxyapatite, whitlockite) was easily determined from the laboratory derived data even when a mixed phase was apparent. Lattice parameter values from the laboratory experiments agreed well with the corresponding synchrotron values and, critically, were determined to precisions that were significantly greater than required for potential clinical exploitation. Conclusion: It has been shown that crystallographic characteristics of microcalcifications can be determined in the laboratory with sufficient precision to have potential clinical value. The work will thus enable exploitation acceleration of these latent microcalcification features as current dependence upon access to limited synchrotron resources is minimized.

Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer.

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 µm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Breast calcification micromorphology classification.

OBJECTIVES: The importance of consistent terminology in describing the appearance of breast calcifications in mammography is well recognised. Imaging of calcifications using electron microscopy is a globally growing field of research. We therefore suggest that the time is ripe to develop a lexicon of terms for classifying the micromorphology of breast calcifications. METHODS: Calcifications within a wide range of histological sections of breast tissue, both benign and malignant, were imaged by Scanning Electron Microscopy (SEM). These images were examined, and the micromorphology of calcifications present was grouped to create a classification system. RESULTS: Based on the appearance of the calcifications observed, we propose five main categories for classification of the micromorphology of breast calcifications, namely, Dense Homogenous, Punctulate, Banded, Spongy and Aggregate. CONCLUSIONS: Use of the descriptive categories outlined here will help to ensure consistency and comparability of published observations on the micromorphology of breast calcifications. ADVANCES IN KNOWLEDGE: This is the first time a lexicon and classification system has been proposed for the micromorphology of breast calcifications, as observed by scanning electron microscopy of histological sections. This will facilitate comparability of observed relationships between micromorphology, mammographic appearance, chemistry and pathology.

Investigating pre-analytical requirements for serum and plasma based infrared spectro-diagnostic.

Infrared spectroscopy is a rapid, easy-to-operate, label-free and therefore cost-effective technique. Many studies performed on biofluids (eg, serum, plasma, urine, sputum, bile and cerebrospinal fluid) have demonstrated its promising application as a clinical diagnostic tool. Given all these characteristics, infrared spectroscopy appears to be an ideal candidate to be implemented into the clinics. However, before considering its translation, a clear effort is needed to standardise protocols for biofluid spectroscopic analysis. To reach this goal, careful investigations to identify and track errors that can occur during the pre-analytical phase is a crucial step. Here, we report for the first time, results of investigations into pre-analytical factors that can affect the quality of the spectral data acquired on serum and plasma, such as the impact of long-term freezing time storage of samples as well as the month-to-month reproducibility of the spectroscopic analysis. The spectral data discrimination has revealed to be majorly impacted by a residual water content variation in serum and plasma dried samples.

Upconversion raster scanning microscope for long-wavelength infrared imaging of breast cancer microcalcifications.

Long-wavelength identification of microcalcifications in breast cancer tissue is demonstrated using a novel upconversion raster scanning microscope. The system consists of quantum cascade lasers (QCL) for illumination and an upconversion system for efficient, high-speed detection using a silicon detector. Absorbance spectra and images of regions of ductal carcinoma in situ (DCIS) from the breast have been acquired using both upconversion and Fourier-transform infrared (FTIR) systems. The spectral images are compared and good agreement is found between the upconversion and the FTIR systems.