Mid-infrared spectroscopy as a tool to monitor molecular changes in fens upon restoration

Dynamic oscillatory experiments and front-face synchronous fluorescence spectroscopy and mid-infrared (mid-IR) spectroscopy have been used to investigate structure evolution, at the macroscopic and molecular levels, during milk acidification kinetics. The studies were performed using skim milk, at two different temperatures (30 °C and 40 °C), to which was added glucono-δ-lactone (GDL) to generate different structural changes in casein micelles and gels. Synchronous fluorescence spectra were recorded in the 250-500 nm excitation wavelength range using an offset of 80 nm between the excitation and emission monochromators for each system during the 300 min acidification kinetics. The change in the fluorescence intensity at 281 nm reflects the pH-induced physicochemical changes of casein micelles and, in particular, structural changes in the micelles in the pH range 5.5-5.0. Regarding mid-infrared spectroscopy, the region located between 1700 and 1500 cm(-1), corresponding to the amide I and II bands, and the 1500-900 cm(-1) region, called the fingerprint region, were considered for the characterization of milk coagulation kinetics. Changes in the absorbance at 1063 cm(-1) as a function of pH for kinetics recorded at 30 °C and 40 °C reflected pH-induced phosphate dissolution in the pH range 5.5-5.0. Compared to rheometry, which reveals microstructure changes only in the gel state, spectroscopic methods make it possible to monitor molecular structure changes in micelles throughout the acidification processes.

Introduction: Microcalcifications are a common feature in mammographic detection of ductal carcinoma in situ (DCIS), and occur in >80% of cases. Known to be present as type I (calcium oxalate-CaO) and type II (carbonated calcium hydroxyapatite-CHAP) microcalcifications, their association with DCIS and their role in the progression of DCIS to invasive breast cancer (IBC) remains unexplored. In an effort to understand the factors involved in DCIS prognosis, it is hypothesized that changes in the chemical composition of calcifications, in tandem with molecular changes in the surrounding soft tissue, will define patients with DCIS who will progress to develop IBC from those who remain with a stable DCIS phenotype. To this end, a novel label-free approach of hyperspectral imaging using mid-infrared (mid-IR) and Raman spectroscopy was used to probe calcification chemistry and molecular composition of the surrounding ductal and stromal soft tissue. The main aim of the work is to identify biomarkers for DCIS prognosis, based on chemical and molecular compositional changes of calcifications and the surrounding soft tissue. It is anticipated that the spectral biomarkers will provide patients and clinicians an informed risk assessment whether to undertake treatment for DCIS or to be placed under active surveillance. Methods: Tissue samples from 422 patient have been obtained and include (i) ‘pure DCIS’ (DCIS without recurrence) (n=193), (ii) ‘DCIS with invasive recurrence’ (DCIS from patients who subsequently were known to develop invasive disease) (n=123), (iii) ‘DCIS plus contemporaneous invasive cancer’ (n=44) and ‘benign’ (n=62) samples. Serial tissue sections were measured using mid-IR and Raman hyperspectral imaging approaches targeting the same calcification and soft tissue regions from specific DCIS ducts. Hyperspectral imaging data was initially pre-processed to digitally remove paraffin and unintended spectral interferences. The pre-processed data was subjected to cluster analysis followed by unsupervised and supervised machine learning classification models to identify spectral features associated with DCIS and its progression to IBC. Results: Cluster analysis based segmentation of hyperspectral images revealed histopathological features including calcifications, epithelium, necrotic areas, connective tissue and stroma. Spectra were extracted from each of the histopathological features using image coordinates, and biomodelling analysis was performed. Initial analysis of 314 calcification images from 170 patients with (i) ‘pure DCIS’ (n=118) and (ii) ‘DCIS with invasive recurrence’ (n=52) showed an area under the receiver operating characteristic (AUROC) mean value of 85% in distinguishing pure DCIS from DCIS that later recurred as IBC. The calcification features appeared to indicate pathology specific changes in phosphate and carbonate content as well as changes in magnesium whitlockite content. Similar analysis of the surrounding soft tissue spectral features showed an AUROC mean value of 85% (necrotic regions surrounding calcifications) and 76% (epithelium) respectively. The epithelial features showed changes in protein secondary structure and content, which together with the calcification changes indicate structural remodelling in DCIS that progresses to IBC, from those that do not. Perspectives: In the ongoing analyses of imaging data from 422 patients, it is anticipated that molecular/structural features from calcification and soft tissue imaging data will provide important cues in understanding DCIS prognosis and could be a promising way forward in determining management of DCIS risk and treatment underpinned by the identification of specific discriminatory spectral markers. Acknowledgments: This work was supported by Cancer Research UK and by KWF Kankerbestrijding (ref. C38317/A24043). Citation Format: Jayakrupakar Nallala, Doriana Calabrese, Sarah Gosling, Esther Lips, Rachel Factor, Allison Hall, Sarah E. Pinder, Ihssane Bouybayoune, Lorraine King, Jeffrey Marks, Thomas Lynch, Donna Pinto, Jelle Wesseling, E Shelley Hwang, Keith Rogers, Nick Stone. Breast microcalcification chemistry predicts DCIS prognosis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-22.

This case study evaluated the efficacy of mid-infrared spectroscopy on the identification of oral squamous cell carcinoma, following the assessment of unstimulated whole saliva. The trial follows a matched case-control design. Saliva samples were characterized through mid-infrared spectroscopy, and chemometric tools were applied to distinguish between case and control participants, further identifying the spectral regions that played a pivotal role in the successful identification of oral squamous cell carcinoma. Mid-infrared spectroscopy was capable to discriminate between cancer patients and matched controls with 100% of correct predictions. Additionally, the spectral regions mostly contributing to the successful prediction were identified and found to be potentially associated with significant molecular changes crucial to the carcinogenic process. The application of mid-infrared spectroscopy in saliva analysis may be regarded as an innovative, noninvasive, low cost, and sensitive technique contributing to the identification of oral squamous cell carcionma.

Near infrared spectroscopic imaging assessment of cartilage composition: Validation with mid infrared imaging spectroscopy

Dual-mode mid-infrared plasmonic metasurface for real-time label-free analysis of live cells.

The chain conformation and hydration structure of an aqueous solution of linear poly(ethylene imine) (LPEI) have been studied by the use of mid-infrared (mid-IR) and near-infrared (NIR) spectrometries to explore the molecular mechanism of an upper critical solution temperature (UCST)-type solubility change. When LPEI was dissolved in water, the solution became a gel at room temperature. Mid-IR and NIR absorption bands of the LPEI gel appeared at the same positions as the bands of the dihydrate crystal of LPEI in a film, which suggested that LPEI formed dihydrate crystallite in water at room temperature. This means that LPEI is in a planar-zigzag structure even in water at room temperature. When the gel was heated, it was changed to a transparent aqueous solution. Both mid-IR and NIR bands of an LPEI aqueous solution above 64 degrees C exhibited a broader bandwidth than those at room temperature. Judging from the change of the bandwidth, the LPEI chain in water was disordered by heating to take a random-coil form. In addition, band locations of the CH stretching first-overtone vibration bands in a NIR spectrum of the solution above 64 degrees C were higher than those of melted dry LPEI, which was reasonably understandable that the disordered LPEI chains in the aqueous solution were in a highly hydrated state. These spectral results have revealed that the change from the planar-zigzag form to the random-coil one in water is a trigger to begin the UCST-like solubility change on heating followed by hydration.

Monitoring of alcoholic fermentation using near infrared and mid infrared spectroscopies combined with electronic nose and electronic tongue

The aim of the present study is to evaluate the potential of mid-infrared spectroscopy in combination with two-dimensional correlation spectroscopy (2DCOS) and partial least squares discriminant analysis (PLS-DA) to monitor molecular structure evolution of milk mixtures of two species (cow and camel) during enzymatic coagulation. Molecular structure changes and texture characteristics were investigated on five different milk formulations by mixing camel milk (CaM) and cow milk (CM) (i.e., CaM, CaM3:CM1, CaM1:CM1, CaM1:CM3, and CM, (v/v)). Regarding MIR spectroscopy, two spectral regions were considered to monitor milk coagulation, the fatty acid acyl-chain region (3000–2800 cm−1) and the protein region (1700–1500 cm−1). Different formulation dissimilarities were revealed through the synchronous 2DCOS spectra and their respective auto-peaks. Moreover, using the cross-peak symbols of the synchronous and asynchronous spectra helps to establish the sequence of molecular structure modifications during coagulation. Finally, the analysis of the 2DCOS-MIR synchronous and asynchronous maps by PLS-DA gave good discriminant accuracy (88 to 100%) between each formulation. Those results highlighted that different synchronous and asynchronous chemical phenomena occurred during milk coagulation depending on the milk formulation. Texture attributes were influenced by camel milk ratio in mixtures based on the initial composition of milks. Samples containing more than 50% of CM showed significantly higher hardness compared to pure CaM and CaM3:CM; however, CM gel displayed lower springiness contrary to CaM. Textural characteristics of the resulted gels also confirmed that 2DCOS MIR observations are consistent with textural observations of the gels obtained after milk mixtures coagulation.

Molecular characterization of green lentil flours using synchrotron X-rays and Fourier transform mid-infrared techniques

Comparative analysis of different transformed Saccharomyces cerevisiae strains based on high-throughput Fourier transform infrared spectroscopy

Study question Can we develop a real-time diagnostic tool for chronic endometritis (CE) by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to evaluate biopsies obtained during hysteroscopy? Summary answer A discrimination model based on the absorbance data was developed by machine learning techniques, differentiating between positive and negative CE histopathology with 97% accuracy. What is known already CE is diagnosed in approximately 15% of infertile women who undergo in vitro fertilization (IVF), in 42% of women with recurrent implantation failure (RIF), and in 57.8% of women with RPL. Diagnosis is done by endometrial biopsy, and the presence of plasma cells in the endometrial stroma is the generally accepted histological diagnostic criterion. However, the histological detection of CE is time-consuming and difficult. ATR-FTIR spectroscopy is a non-destructive method that can provide valuable information on biochemical changes that occur during pathological processes, such as inflammation and cancer. Study design, size, duration We performed a prospective study in which fresh biopsies of endometrium were obtained during standard hysteroscopies. Each biopsy was examined by the spectrophotometer and afterward by histopathological analysis in which multiple myeloma oncogene 1 (MUM–1) staining for plasma cells, a marker of CE, was performed. We planned to investigate 80 samples to develop a discrimination model, and another 40 samples for validation of the model. The study was planned to last two years. Participants/materials, setting, methods Women that underwent hysteroscopy as a part of infertility evaluation were recruited. The hysteroscopies and the biopsy evaluation were performed at the same center. A cut-off of 8 MUM–1 positive cells per 10 high power fields (HPF) was set. We compared the spectroscopy analysis of the positive CE group (≥8) and the negative CE group (<8). Machine learning technique was utilized to build discrimination models. Data analysis was performed using Matlab and Unscrambler software packages. Main results and the role of chance We present preliminary results for our study. Forty-two women were recruited from January 2020 until November 2020. Of the 42 measured spectra, three were discarded due to high measurement noise. Of the 39 biopsies, 33 had MUM–1<8 (CE negative group) and 6 had MUM–1≥8 (CE positive group). Measured spectra of tissue smears from CE negative and positive groups differed from each other in the spectral range of 850–990 [cm–1] (p < 0.05). This wavenumber can be associated with the C-H in-plane bend in the alkene group (CnH2n). A discriminant model was developed between the groups using the Principal Component Analysis and Linear Discriminant Analysis techniques. The accuracy obtained by the model was 97%. We divided the 39 hysteroscopies based on the CE signs into 2 groups: “Negative hysteroscopic-CE” and “Positive hysteroscopic-CE”. Positive hysteroscopic signs were micropolyps, strawberry pattern, hyperemia, punctuation, or pale endometrium. Twenty-three samples were taken in the Negative group and 16 samples were taken in the Positive group. However, measured spectra of tissue smears from negative and positive hysteroscopy groups were not significantly different. The correlation coefficient between hysteroscopy groups and MUM–1 score was r = 0.29, meaning that the characteristic signs of CE in hysteroscopy were not correlated to the histopathology. Limitations, reasons for caution First, these are preliminary results and we need to investigate more samples to validate our model. Second, diagnostic criteria for CE are diverse in the literature and we chose 8 MUM–1 positive cells in 10 HPF, a criterion which may not be accepted by all experts in the field. Wider implications of the findings: ATR-FTIR spectroscopy is highly sensitive to molecular changes and has been utilized as a diagnostic tool in a variety of clinical studies. While histopathological results take about two weeks, ATR-FTIR spectroscopy might give us the possibility to diagnose CE in real-time, allowing an immediate initiation of the appropriate treatment. Trial registration number ClinicalTrials.gov Identifier: NCT04197167