Multimodal Vibrational Studies of Drug Uptake In Vitro: Is the Whole Greater than the Sum of Their Parts?

Herein, we investigated the use of multimodal Raman and infrared (IR) spectroscopic microscopy for the elucidation of drug uptake and subsequent cellular responses. Firstly, we compared different methods for the analysis of the combined data. Secondly, we evaluated whether the combined analysis provided enough benefits to justify the fusion of the data. A459 cells inoculated with doxorubicin (DOX) at different times were fixed and analysed using each technique. Raman spectroscopy provided high sensitivity to DOX and enabled an accurate estimation of the drug uptake at each time point, whereas IR provided a better insight into the resultant changes in the biochemical composition of the cell. In terms of benefits of data fusion, 2D correlation analysis allowed the study of the relationship between IR and Raman variables, whereas the joint analysis of IR and Raman enabled the correlation of the different variables to be monitored over time. In summary, the complementary nature of IR and Raman makes the combination of these vibrational techniques an appealing tool to follow drug kinetics and cellular response. Funding information: H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: Spectro-metrics- 020-MSCA-IF-2017 Project ID:79628; National Science Centre, Grant/Award Number: UMO 2016/23/B/NZ4/0137

Towards the Point of Care and Noninvasive Classification of Bladder Cancer from Urine Sediment Infrared Spectroscopy. Spectral differentiation of normal, abnormal and cancer patients

Bladder cancer (BC) is the 9th cancer cause of death and one of most cost-intensive in the world. The diagnostic tools are still not at all satisfactory. Herein we evaluated the potential of infrared spectroscopy to detect molecular changes that precede and accompany the carcinogenesis in voided urine sediment. We collected 165 samples from patients being diagnosed for BC and measured them with attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FTIR). Samples were primarily divided into three groups according to cytology that indicated the presence of normal, abnormal and cancer cells. ATR FTIR spectra of sediments were analyzed with the use of partial least square discriminant analysis (PLSDA). The 1800–750 cm− 1 region discriminated the three groups with selectivity and sensitivity values around 68% using cytology as a reference method. These cross-validation values (which were found significant according to a permutation test) were comparable to the sensitivity and specificity values of cytology versus the gold standard (histology). The average spectra of each class and the regression vectors of the PLS-DA indicated that an increased content of carbohydrates and nucleic acids as well as transformations of protein secondary structures were the main discriminators of healthy patients from abnormal and cancer groups. Additionally, we revised the obtained classification according to diagnosis made on histopathological assessment of bladder sections. We finally discuss the potential of the technique to be used as a Point of Care (PoC) testing tool

Eosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy

Leukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process

SERS-based monitoring of the intracellular pH in endothelial cells:the influence of the extracellular environment and tumour necrosis factor-alpha

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-alpha (TNF alpha). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments.This work was supported by the National Center of Science (grant PRELUDIUM DEC-2012/05/N/ST4/00218) and by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, no. POIG.01.01.02-00-069/09). We also thank the University of Edinburgh School of Chemistry for the Neil Campbell Travel Award for supporting LJ. We also thank Joanna Jalmuzna from the Department of Mathematics and Computer Sciences, Jagiellonian University in Krakow for fitting the calibration curve using Gnuplot software

FTIR spectroscopic imaging supports urine cytology for classification of low- and high-grade bladder carcinoma

SIMPLE SUMMARY: Human urine cytological samples were investigated using Fourier transform infrared spectroscopic imaging in terms of recognition of bladder cancer. The clustering of IR spectra of whole cytological smears revealed very good spectral correlation with normal urothelial cell features. Next, the combination of spectral information derived from unsupervised hierarchical cluster analysis and partial least square discriminant analysis (PLS-DA) classified normal vs. low- and high-grade bladder urothelial carcinoma with sensitivity and specificity of 90–97%. ABSTRACT: Bladder urothelial carcinoma (BC) is a common, recurrent, life-threatening, and unpredictable disease which is difficult to diagnose. These features make it one of the costliest malignancies. Although many possible diagnostic methods are available, molecular heterogeneity and difficulties in cytological or histological examination induce an urgent need to improve diagnostic techniques. Herein, we applied Fourier transform infrared spectroscopy in imaging mode (FTIR) to investigate patients’ cytology samples assigned to normal (N), low-grade (LG) and high-grade (HG) BC. With unsupervised hierarchical cluster analysis (UHCA) and hematoxylin-eosin (HE) staining, we observed a correlation between N cell types and morphology. High-glycogen superficial (umbrella) and low-glycogen piriform urothelial cells, both with normal morphology, were observed. Based on the spectra derived from UHCA, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed, indicating a variation of protein content between the patient groups. Moreover, BC spectral cytology identified a low number of high-glycogen cells for which a shift of the carbohydrate/phosphate bands was also observed. Despite high cellular heterogeneity, PLS-DA was able to classify the spectra obtained. The voided urine FTIR cytology is one of the options that might be helpful in BC diagnosis, as high sensitivity and specificity up to 97% were determined

pH and Substrate Effect on Adsorption of Peptides Containing <i>Z</i> and <i>E</i> Dehydrophenylalanine. Surface-Enhanced Raman Spectroscopy Studies on Ag Nanocolloids and Electrodes

The silver substrates and pH dependent surface-enhanced Raman scattering (SERS) spectra of unsaturated derivatives of di- and tripeptides (dehydropeptides) are investigated. Experimental spectra were interpreted with the help of DFT calculations and normal-mode analysis. We choose as objects of our studies modified but natural peptides containing <i>Z</i> or <i>E</i> dehydrophenylalanine (^(Z)/(E)ΔPhe) residue to study an effect of the type of the isomer on the interaction between the peptide and silver surfaces in the form of nanocolloidal particles and an electrochemically roughened electrode. We also observed that the SERS profile is sensitive to both the type of the studied SERS active substrate and pH, especially for the adsorption on the silver colloid. In general, all dehydropeptides interact with both SERS substrates upon deprotonation of the C-end of the molecule. The participation of the other fragments of the adsorbates such as the N-terminal amino group and the dehydroresidue is also manifested in the SERS spectra. Their orientation with respect to the silver surfaces is discussed in detail

Evaluation of grade and invasiveness of bladder urothelial carcinoma using infrared imaging and machine learning

Urothelial bladder carcinoma (BC) is primarily diagnosed with a subjective examination of biopsies by histopathologists, but accurate diagnosis remains time-consuming and of low diagnostic accuracy, especially for low grade non-invasive BC. We propose a novel approach for high-throughput BC evaluation by combining infrared (IR) microscopy of bladder sections with machine learning (partial least squares-discriminant analysis) to provide an automated prediction of the presence of cancer, invasiveness and grade. Cystoscopic biopsies from 50 patients with clinical suspicion of BC were histologically examined to assign grades and stages. Adjacent tissue cross-sections were IR imaged to provide hyperspectral datasets and cluster analysis segregated IR images to extract the average spectra of epithelial and subepithelial tissues. Discriminant models, which were validated using repeated random sampling double cross-validation, showed sensitivities (AUROC) ca. 85% (0.85) for the identification of cancer in epithelium and subepithelium. The diagnosis of non-invasive and invasive cases showed sensitivity values around 80% (0.84-0.85) and 76% (0.73-0.80), respectively, while the identification of low and high grade BC showed higher sensitivity values 87-88% (0.91-0.92). Finally, models for the discrimination between cancers with different invasiveness and grades showed more modest AUROC values (0.67-0.72). This proves the high potential of IR imaging in the development of ancillary platforms to screen bladder biopsies

FTIR Spectroscopic Imaging Supports Urine Cytology for Classification of Low- and High-Grade Bladder Carcinoma

Bladder urothelial carcinoma (BC) is a common, recurrent, life-threatening, and unpredictable disease which is difficult to diagnose. These features make it one of the costliest malignancies. Although many possible diagnostic methods are available, molecular heterogeneity and difficulties in cytological or histological examination induce an urgent need to improve diagnostic techniques. Herein, we applied Fourier transform infrared spectroscopy in imaging mode (FTIR) to investigate patients’ cytology samples assigned to normal (N), low-grade (LG) and high-grade (HG) BC. With unsupervised hierarchical cluster analysis (UHCA) and hematoxylin-eosin (HE) staining, we observed a correlation between N cell types and morphology. High-glycogen superficial (umbrella) and low-glycogen piriform urothelial cells, both with normal morphology, were observed. Based on the spectra derived from UHCA, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed, indicating a variation of protein content between the patient groups. Moreover, BC spectral cytology identified a low number of high-glycogen cells for which a shift of the carbohydrate/phosphate bands was also observed. Despite high cellular heterogeneity, PLS-DA was able to classify the spectra obtained. The voided urine FTIR cytology is one of the options that might be helpful in BC diagnosis, as high sensitivity and specificity up to 97% were determined

Comparative Studies on IR, Raman, and Surface Enhanced Raman Scattering Spectroscopy of Dipeptides Containing ΔAla and ΔPhe

Three dipeptides containing dehydroresidues (ΔAla, Δ^(<i>Z</i>)Phe, and Δ^(<i>E</i>)Phe) were examined by IR, Raman, and surface-enhanced Raman techniques for the first time. The effect of the size and isomer type of the β-substituent in the dehydroresidue on the conformational structure of the peptide was evaluated by using the analysis of IR and Raman bands. Additionally, SERS spectroscopy provided insight into the adsorption mechanism of these species on the metal surface. SERS spectra were recorded at alkaline pH on the silver sol using visible light excitation. The dehydroresidues studied here strongly influenced the SERS profile of the peptides. The most pronounced SERS signal for all dipeptides was assigned to the symmetric stretching vibration of the carboxylate ions. This indicates that the dehydropeptides studied here primarily adsorb via the deprotonated carboxylic group. Additionally, the enhanced SERS bands in the range 1550–1650 cm^–1 show differences in contribution of the dehydroresidue to the adsorption mechanism of the studied peptides

Multimodal vibrational studies of drug uptake in vitro: Is the whole greater than the sum of their parts?

Herein, we investigated the use of multimodal Raman and infrared (IR) spectroscopic microscopy for the elucidation of drug uptake and subsequent cellular responses. Firstly, we compared different methods for the analysis of the combined data. Secondly, we evaluated whether the combined analysis provided enough benefits to justify the fusion of the data. A459 cells inoculated with doxorubicin (DOX) at different times were fixed and analysed using each technique. Raman spectroscopy provided high sensitivity to DOX and enabled an accurate estimation of the drug uptake at each time point, whereas IR provided a better insight into the resultant changes in the biochemical composition of the cell. In terms of benefits of data fusion, 2D correlation analysis allowed the study of the relationship between IR and Raman variables, whereas the joint analysis of IR and Raman enabled the correlation of the different variables to be monitored over time. In summary, the complementary nature of IR and Raman makes the combination of these vibrational techniques an appealing tool to follow drug kinetics and cellular response. Funding information: H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: Spectro-metrics- 020-MSCA-IF-2017 Project ID:79628; National Science Centre, Grant/Award Number: UMO 2016/23/B/NZ4/0137