Infrared and 2-Dimensional Correlation Spectroscopy Study of the Effect of CH3NH3PbI3 and CH3NH3SnI3 Photovoltaic Perovskites on Eukaryotic Cells.

We studied the effect of the exposure of human A549 and SH-SY5Y cell lines to aqueous solutions of organic/inorganic halide perovskites CH3NH3PbI3 (MAPbI3) and CH3NH3SnI3 (MASnI3) at the molecular level by using Fourier transform infrared microspectroscopy. We monitored the infrared spectra of some cells over a few days following exposure to the metals and observed the spectroscopic changes dominated by the appearance of a strong band at 1627 cm-1. We used Infrared (IR) mapping to show that this change was associated with the cell itself or the cellular membrane. It is unclear whether the appearance of the 1627 cm-1 band and heavy metal exposure are related by a direct causal relationship. The spectroscopic response of exposure to MAPbI3 and MASnI3 was similar, indicating that it may arise from a general cellular response to stressful environmental conditions. We used 2D correlation spectroscopy (2DCOS) analysis to interpret spectroscopic changes. In a novel application of the method, we demonstrated the viability of 2DCOS for band assignment in spatially resolved spectra. We assigned the 1627 cm-1 band to the accumulation of an abundant amide or amine containing compound, while ruling out other hypotheses. We propose a few tentative assignments to specific biomolecules or classes of biomolecules, although additional biochemical characterization will be necessary to confirm such assignments.

Infrared spectra of micro-structured samples with microPhotoacoustic spectroscopy and synchrotron radiation.

Photoacoustic spectroscopy (PAS) measures the photon absorption spectrum of a sample through detection of the acoustic wave generated by the photothermal effect as one modulates the intensity of the incident radiation at each wavelength. We have recently demonstrated the implementation of PAS in a microscopy configuration with mid-infrared radiation (microPAS). In the present work, we describe the performance of microPAS using synchrotron radiation (SR) in diffraction-limited spectromicroscopy and imaging experiments. Spectra were obtained for polystyrene beads, polypropylene fibres, and single fibres of human hair. SR produced microPAS spectra of much higher intensity as compared with those obtained using conventional mid- and near-infrared sources. For hair samples, the penetration depth of mid-infrared light, even with bright SR, is significantly shorter than the probed sample thickness at very low modulation frequencies resulting in saturated PAS spectra. In contrast, microPAS spectra of polymer beads were in general of much better quality than those obtained with conventional sources. We also demonstrated the capability to collect line profiles and line spectra at diffraction limited spatial resolution. The microPAS spectra of beads appear free from appreciable bandshape distortions arising from the real part of the refractive index of the sample. This observation confirms microPAS as an absorption-only technique and establishes it as a valuable new tool in the microspectroscopic analysis of particulates and of samples with a complex topography.

Mid-infrared spectroscopy and microscopy of subcellular structures in eukaryotic cells with atomic force microscopy - infrared spectroscopy.

Atomic force microscopy - infrared (AFM-IR) spectroscopy allows spectroscopic studies in the mid-infrared (mid-IR) spectral region with a spatial resolution better than is allowed by the diffraction limit. We show that the high spatial resolution can be used to perform spectroscopic and imaging studies at the subcellular level in fixed eukaryotic cells. We collect AFM-IR images of subcellular structures that include lipid droplets, vesicles and cytoskeletal filaments, by relying on the intrinsic contrast from IR light absorption. We also obtain AFM-IR absorption spectra of individual subcellular structures. Most spectra show features that are recognizable in the IR absorption spectra of cells and tissue obtained with FTIR technology, including absorption bands characteristic of phospholipids and polypeptides. The quality of the spectra and of the images opens the way to structure and composition studies at the subcellular level using mid-IR absorption spectroscopy.

Infrared imaging of small molecules in living cells: from in vitro metabolic analysis to cytopathology.

A major topic in InfraRed (IR) spectroscopic studies of living cells is the complexity of the vibrational spectra, involving hundreds of overlapping absorption bands from all the cellular components present at detectable concentrations. We focus on the relative contribution of both small-molecule metabolites and macromolecules, while defining the spectroscopic properties of cells and tissue in the middle IR (midIR) region. As a consequence, we show the limitations of current interpretative schemes that rely on a small number of macromolecules for IR band assignment. The discussion is framed specifically around the glycolytic metabolism of cancer cells because of the potential pharmacological applications. Several metabolites involved in glycolysis by A549 lung cancer cells can be identified by this approach, which we refer to as Correlated Cellular Spectro-Microscopy (CSM). It is noteworthy that the rate of formation or consumption of specific molecules could be quantitatively assessed by this approach. We now extend this analysis to the two-dimensional case by performing IR imaging on single cells and cell clusters, detecting variations of metabolite concentration in time and space across the sample. The molecular detail obtained from this analysis allows its use in evaluating the pharmacological effect of inhibitors of glycolytic enzymes with potential consequences for in vitro drug testing. Finally we highlight the implications of the spectral contribution from cellular metabolites on applications in IR spectral cytopathology (SCP).

Synthesis, characterization and cellular location of cytotoxic constitutional organometallic isomers of rhenium delivered on a cyanocobalmin scaffold.

Constitutional isomers of cyanocobalamin adducts based on a fluorescent rhenium tris-carbonyl diimine complex were prepared, characterized and tested against PC-3 cancer cells. The adducts differ only in the relative binding position of the organometallic species which is either bound at the cyano or the 5'-hydroxo group of vitamin B12. When tested for their cytotoxic potency, the species showed IC50 values in the low µM rage. Upon conjugation to the vitamin an energy transfer process causes an extremely low quantum yield of fluorescence emission, making the conjugates unsuitable for fluorescence imaging. However, by exploiting the vibrational signature of the fac-[Re(CO)3](+) core, their cellular distribution was evaluated via FTIR spectromicroscopy.

Real-time metabolic analysis of living cancer cells with correlated cellular spectro-microscopy.

In recent years, major efforts have been devoted to the application of microscopy with mid-infrared light to the study of living cells and tissue. Despite this interest, infrared (IR) microscopy has not realized its full potential in the molecular characterization of living systems. This is partly due to the fact that current approaches for data mining and analysis of IR absorption spectra have not evolved comparably to measurement technology and are not up to the interpretation of the complex spectra of living systems such as cells and tissue. In this work we show that the use of two-dimensional correlation spectroscopy coupled to IR absorption spectro-microscopy allows us to extract the spectral components of individual metabolites from time-resolved IR spectra of living cells. We call this method correlated cellular spectro-microscopy, and we implement it in the study of the glycolytic metabolism of cancer cells. We show that the method can detect intermediates of the glycolytic pathway, quantify their rate of formation, and correlate this with variations in pH, all in a single measurement. We propose the method as a useful tool for the quantitative description of metabolic processes in living cells and for the validation of drug candidates aimed at suppressing glycolysis in cancer cells.

Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women: implications for latent stages of breast carcinogenesis.

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States' high risk-posing environment and: (2) marked (> 1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE- cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the "biochemical fingerprint" of 4HNE+ vs. 4HNE- luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy's ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

Identification and characterization of stemlike cells in human esophageal adenocarcinoma and normal epithelial cell lines.

OBJECTIVE: Recent studies have suggested that human solid tumors may contain subpopulations of cancer stem cells with the capacity for self-renewal and the potential to initiate and maintain tumor growth. The aim of this study was to use human esophageal cell lines to identify and characterize putative esophageal cancer stem cell populations. METHODS: To enrich stemlike cells, Het-1A (derived from immortalized normal esophageal epithelium), OE33, and JH-EsoAd1 (each derived from primary esophageal adenocarcinomas) were cultured using serum-free media to form spheres. A comprehensive analysis of parent and spheroid cells was performed by flow cytometry, Western blot analysis, immunohistochemistry and polymerase chain reaction array to study cancer stem cell-related genes, colony formation assays to assess clonogenicity, xenotransplantation to assess tumorigenicity, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays to assess chemosensitivity to 5-fluorouracil and cisplatin. RESULTS: For all cell lines, clonogenicity, tumorigenicity, and chemoresistance to 5-fluorouracil and cisplatin were significantly higher than for spheroid cells compared with parent cells. Spheroids exhibited an increased frequency of cells expressing integrin α6(bri)/CD71(dim), and Achaete-scute complex homolog 2 messenger RNA and protein were also significantly overexpressed in spheroid cells compared with parent cells. CONCLUSIONS: The higher clonogenicity, tumorigenicity, and drug resistance exhibited by spheroids derived from Het-1A, OE33, and JH-EsoAd1 reflects an enrichment of stemlike cell populations within each esophageal cell line. Esophageal cells enriched for integrin α6(bri)/CD71(dim) and/or overexpressing Achaete-scute complex homolog 2 would appear to represent at least a subpopulation of stemlike cells in Het-1A, OE33, and JH-EsoAd1.

Characterization of Barrett esophagus and esophageal adenocarcinoma by Fourier-transform infrared microscopy.

The objective of this exploratory study was to evaluate the feasibility of using Fourier-Transform Infrared (FTIR) spectromicroscopy to characterize formalin-fixed, paraffin-embedded human esophageal tissues. Matched histologically normal esophageal squamous epithelium (NS), premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC) tissues, each defined according to strict clinicopathologic criteria, were obtained from patients who underwent esophageal resection. Using confocal IR microscopy, measurements in the mid-IR spectral region were carried out in transflection configuration, scanning regions of interest in 15 microm steps. A multidimensional dataset reporting the spectroscopic properties at each sampled point were analyzed by performing a hierarchical cluster analysis on the second derivative of spectral traces. Normal esophageal epithelia were characterized by a few well defined regions, mostly of large size (tens of contiguous pixels), which correlated with tissue histology, specifically the basal cell layer. BE tissues had characteristic regions localized to gland crypts, ranging in size from one pixel to a few tens of pixels, which displayed IR spectra with defined absorption features characteristic of glycoproteins. The incorporation of synchrotron light to improve the resolution of individual cells in BE tissues has demonstrated that these glycoproteins are associated with goblet cells, the characteristic cell type defining BE. Whereas the highly fragmented regions identified in EADC likely reflect tumor heterogeneity, FTIR mapping would appear to be a potentially useful technique to identify premalignant BE tissues. The technical feasibility of using FTIR to characterize formalin-fixed, paraffin-embedded human esophageal tissues demonstrates the potential of this technique to study archival human BE tissue specimens via automated screening techniques.