Novel infrared and Raman spectroscopic imaging for the elucidation of specific changes in breast microcalcifications

Breast cancer is the second most common cause of death from cancer in women, accounting for more than 1 million deaths globally per year. Current detection is based on X-ray mammographic screening, which involves the use of ionising radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. The presence of microcalcifications in breast tissue has been associated with malignant disease. Unfortunately, X-ray mammography and MRI scanning techniques are not able to discriminate between microcalcifications from a benign lesion and those from a malignant lesion. The aim of this project was to use optical techniques based on vibrational spectroscopy, such as Fourier Transform Infrared (FTIR) absorption and Raman scattering, which are non-destructive, label-free and chemically specific, to investigate the composition of microcalcifications in breast tissue for augmented diagnostics and improved outcome for the patient. This work involved the characterisation of mineral standards of the type that can be found in the breast, in order to identify the precise composition of the microcalcifications. A series of calcium hydroxyapatite (Hap) compounds was used for calibration of the micro-FTIR and Raman spectra. The ratio of carbonate-to-phosphate band intensity for each individual Hap powder was determined and the data were used to assess the level of carbonate substitution in each breast tissue biopsy. In parallel, the analysis of potential precursor mineral phases (namely octacalcium phosphate and amorphous calcium phosphate) revealed similar features to Hap in both FTIR and Raman spectra, which can be translated to the biopsy samples. The accessibility to diverse panels of breast tissue sections (frozen and paraffin-embedded) was a great opportunity to test different approaches. A deparaffinisation protocol was applied to a set of samples for Raman analysis and the process was found not to affect the microcalcification composition. The FTIR analysis of the frozen tissues provided information on the carbonate peak in the short wavelength range (1500-1400 cm-1), which normally contains a strong contribution from paraffin in standard histological specimens. The study of breast tissue sections showed the heterogeneity in composition of microcalcifications between different samples from the same stage of pathology in terms of protein, lipid - which is usually not observed in formalin-fixed paraffin-preserved (FFPE) sections - and carbonate content. The mineralisation of the MDA-MB-231 breast cell line induced by two osteogenic agents: inorganic phosphate (Pi) and -Glycerophosphate (G) was investigated using Raman micro-spectroscopy. The uptake of osteogenic agent induced a faster mineralisation for cells cultured with a medium supplemented in Pi (day 3) than G (day 11). A shift (± 3 cm-1) of the phosphate peak at 956 cm-1 in the Raman spectra was apparent when the culture medium was supplemented with G, indicating the presence of precursor phase (octacalcium phosphate) during Hap crystal formation. New IR technologies such as bright laser sources e.g. quantum cascade laser (QCL) open possibilities for the analysis of biological samples. They allowed us to achieve a better signal-to-noise ratio than Globar thermal sources used in traditional FTIR systems, particularly on optically dense samples such as calcifications. The ability of selecting specific incident wavelengths allows significant improvements in the acquisition time. This work illustrates for the first time the identification of microcalcifications using a QCL source in the long wavelength range coupled to an upconversion system with a silicon detector for efficient sensing. The upconverted images showed a good agreement with the micro-FTIR images. Vibrational spectroscopy has been shown to be a powerful tool for discrimination of mineral species in breast calcification. These techniques can provide complementary information for the pathologist to be able to classify breast pathologies - benign, ductal carcinoma in situ (DCIS) and invasive cancer - with higher accuracy.European Commissio

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

This is the final version. Available from Optical Society of America via the DOI in this record. 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.European Unio

Biofluid infrared spectro-diagnostics : pre-analytical considerations for clinical applications

Several proof-of-concept studies on vibrational spectroscopy of biofluids have demonstrated that the methodology has promising potentials as a clinical diagnostic tool. However, these studies also show that there is lack of standardised protocol in sample handling and preparation prior to spectroscopic analysis. One of the most important sources of analytical errors is the pre-analytical phase. For the technique to be translated into clinics, it is clear that a very strict protocol needs to be established for such biological samples. This study focuses on some of the aspects of the pre-analytical phase in the development of high=throughput Fourier Transform Infrared (FTIR) spectroscopy of some of the most common biofluids such as serum, plasma and bile. Pre-analytical considerations that can impact either the samples (solvents, anti-coagulants, freeze-thaw cycles....) and/or spectroscopic analysis (sample preparation such as drying, deposit methods, volumes. substrates. operators dependence...) and consequently on the quality and the reproducibility of spectral data will be discussed in the report

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 β-glycerophosphate (βG), 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 βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG 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

Assessment of Compressive Raman versus Hyperspectral Raman for Microcalcification Chemical Imaging

International audienceWe experimentally implement a compressive Raman technology (CRT) that incorporates chemometricanalysis 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 EMCCDbased systems, respectively