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We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.

We use photothermal induced resonance (PTIR) imaging and spectroscopy, in resonant and non-resonant mode, to study the cytoplasmic membrane and surface of intact cells. Non-resonant PTIR images apparently provide rich details of the cell surface. However, we show that non-resonant image contrast does not arise from the infrared absorption of surface molecules and is instead dominated by the mechanics of tip-sample contact. In contrast, spectra and images of the cellular surface can be selectively obtained by tuning the pulsing structure of the laser to restrict thermal wave penetration to the surface layer. Resonant PTIR images reveal surface structures and domains that range in size from about 20 nm to 1 μm and are associated with the cytoplasmic membrane and its proximity. Resonant PTIR spectra of the cell surface are qualitatively comparable to far-field IR spectra and provide the first selective measurement of the IR absorption spectrum of the cellular membrane of an intact cell. In resonant PTIR images, signal intensity, and therefore contrast, can be ascribed to a variety of factors, including mechanical, thermodynamic and spectroscopic properties of the cellular surface. While PTIR images are difficult to interpret in terms of spectroscopic absorption, they are easy to collect and provide unique contrast mechanisms without any exogenous labelling. As such they provide a new paradigm in cellular imaging and membrane biology and can be used to address a range of critical questions, from the nature of membrane lipid domains to the mechanism of pathogen infection of a host cell.

Photothermal-induced resonance (PTIR) is increasingly used in the measurement of infrared absorption spectra of submicrometer objects. The technique measures IR absorption spectra by relying on the photothermal effect induced by a rapid pulse of light and the excitation of the resonance spectrum of an AFM cantilever in contact with the sample. In this work, we assess the spatial resolution and depth response of PTIR in resonant mode while systematically varying the pulsing parameters of the excitation laser. We show that resolution is always much better than predicted by existing theoretical models. Higher frequency, longer pulse length, and shorter interval between pulses improve resolution, eventually providing values that are comparable to or even better than tip size. Pulsing parameters also affect the intensity of the signal and the surface selectivity in PTIR images, with higher frequencies providing increased surface selectivity. The observations confirm a difference in signal generation between resonant PTIR and other photothermal techniques that we ascribe to nonlinearity in the PTIR signal. In analogy with optical imaging, we show that PTIR takes advantage of such nonlinearity to perform photothermal measurements that are super-resolved when compared to the resolution allowed by the thermal wavelength. Finally, we show that by controlling the pulsing parameters of the laser we can devise high resolution surface sensitive measurements that do not rely on the use of optical enhancement effects.

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.

We studied the effect of the exposure of human A549 and SH-SY5Y cell lines to aqueous solutions of organic/inorganic halide perovskites CH 3 NH 3 PbI 3 (MAPbI 3 ) and CH 3 NH 3 SnI 3 (MASnI 3 ) 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 MAPbI 3 and MASnI 3 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., Competing Interests: Figure A1. Quantification of living cells upon exposure to MAPbI3 and MASnI3. (a,c) SH-SY5Y neuroblastoma cells; (b,d) A549 lung epithelial cells. Histograms show the average of triplicate measurements. Bars are means ± σ. The histograms show an average of at least three independent repeats. Bars are means ± σ. One-way ANOVA tests followed by Tukey-Kramer post-hoc tests were performed (non-treated vs. MAPbI3 or vs. MASnI3 treated conditions), *p < 0.01, **p < 0.005, ***p < 0.0005. Panel b is reproduced from [2] with permission from the Royal Society of Chemistry. Panels (a–d) are reproduced from [6] (I.B. doctoral thesis).

Photothermal-induced resonance (PTIR) spectroscopy and imaging with infrared light has seen increasing application in the molecular spectroscopy of biological samples. The appeal of the technique lies in its capability to provide information about IR light absorption at a spatial resolution better than that allowed by light diffraction, typically below 100 nm. In the present work, we tested the capability of the technique to perform measurements with subcellular resolution on intact eukaryotic cells, without drying or fixing. We demonstrate the possibility of obtaining PTIR images and spectra from the nucleus and multiple organelles with high resolution, better than that allowed by diffraction with infrared light. We obtain particularly strong signal from bands typically assigned to acyl lipids and proteins. We also show that while a stronger signal is obtained from some subcellular structures, other large subcellular components provide a weaker or undetectable PTIR response. The mechanism that underlies such variability in response is presently unclear. We propose and discuss different possibilities, addressing thermomechanical, geometrical, and electrical properties of the sample and the presence of cellular water, from which the difference in response may arise.

Mid-infrared absorption spectroscopy has been used extensively to study the molecular properties of cell membranes and model systems. Most of these studies have been carried out on macroscopic samples or on samples a few micrometers in size, due to constraints on sensitivity and spatial resolution with conventional instruments that rely on far-field optics. Properties of membranes on the scale of nanometers, such as in-plane heterogeneity, have to date eluded investigation by this technique. In the present work, we demonstrate the capability to study single bilayers of phospholipids with near-field mid-infrared spectroscopy and imaging and achieve a spatial resolution of at least 40 nm, corresponding to a sample size of the order of a thousand molecules. The quality of the data and the observed spectral features are consistent with those reported from measurements of macroscopic samples and allow detailed analysis of molecular properties, including orientation and ordering of phospholipids. The work opens the way to the nanoscale characterization of the biological membranes for which phospholipid bilayers serve as a model.

The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron-sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

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).

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.

We show that attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be used to determine the surface charge density (SCD) of colloidal silica nanoparticles (NPs) in aqueous solution. We identify the Si-O stretch vibrations of neutral surface bound silanol, ≡Si-OH, and of the deprotonated group, ≡Si-O(-). The position of the Si-(OH) stretch vibration is shown to directly correlate with the NPs SCD as determined by traditional potentiometric titrations, shifting to lower wavenumber (cm(-1)) with increasing density of ≡Si-O(-). The origin of this shift is discussed in terms of inductive effects that reduce the ionic character of the Si-(OH) bond after delocalization of the negative charge left on a terminal ≡Si-O(-) group across the atoms within ∼1 nm of the charged site. Using this new methodology, we quantitatively determine the SCD of 9, 14, and 25 nm diameter colloidal silica in varying concentrations of NaCl electrolyte at different bulk pH. This novel spectroscopic approach to investigate SCDs provides several opportunities for in situ coupling, for example, in microfluidic channels or with liquid microjets, and requires only very little sample—all potential advantages over a traditional potentiometric titration.

Twisted stalks are organo-mineral structures produced by some microaerophilic Fe(II)-oxidizing bacteria at O2 concentrations as low as 3 μM. The presence of these structures in rocks having experienced a diagenetic history could indicate microbial Fe(II)-oxidizing activity as well as localized abundance of oxygen at the time of sediment deposition. Here we use spectroscopy and analytical microscopy to evaluate if--and what kind of--transformations occur in twisted stalks through experimental diagenesis. Unique mineral textures appear on stalks as temperature and pressure conditions increase. Haematite and magnetite form from ferrihydrite at 170 °C-120 MPa. Yet the twisted morphology of the stalks, and the organic matrix, mainly composed of long-chain saturated aliphatic compounds, are preserved at 250 °C-140 MPa. Our results suggest that iron minerals might play a role in maintaining the structural and chemical integrity of stalks under diagenetic conditions and provide spectroscopic signatures for the search of ancient life in the rock record.

Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

We describe a novel cell used to combine in situ transmission X-ray absorption spectroscopy (XAS) with diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) in a single experiment. The novelty of the cell design compared to current examples is that both radiations are passed through an X-ray and IR transparent window in direct contact with the sample. This innovative geometry also offers a wide surface for IR collection. In order to avoid interference from the crystalline IR transparent materials (e.g., CaF2, MgF2, diamond) a 500 μm carbon filled hole is laser drilled in the center of a CaF2 window. The cell is designed to represent a plug flow reactor, has reduced dead volume in order to allow for fast exchange of gases and is therefore suitable for experiments under fast transients, e.g., according to the concentration modulation approach. High quality time-resolved XAS and DRIFTS data of a 2 wt.% Pt/Al2O3 catalyst are obtained in concentration modulation experiments where CO (or H2) pulses are alternated to O2 pulses at 150 °C. We show that additional information can be obtained on the Pt redox dynamic under working conditions thanks to the improved sensitivity given by the modulation approach followed by Phase Sensitive Detection (PSD) analysis. It is anticipated that the design of the novel cell is likely suitable for a number of other in situ spectroscopic and diffraction methods.

We successfully tested the viability of using synchrotron-based full-field infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information., (Copyright © 2014. Published by Elsevier B.V.)

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.

Carbon monoxide releasing molecules (CORMs) are an emerging class of pharmaceutical compounds currently evaluated in several preclinical disease models. There is general consensus that the therapeutic effects elicited by the molecules may be directly ascribed to the biological function of the released CO. It remains unclear, however, if cellular internalization of CORMs is a critical event in their therapeutic action. To address the problem of cellular delivery, we have devised a general strategy which entails conjugation of a CO-releasing molecule (here a photoactivated CORM) to the 5'-OH ribose group of vitamin B12. Cyanocobalamin (B12) functions as the biocompatible water-soluble scaffold which actively transports the CORM against a concentration gradient into the cells. The uptake and cellular distribution of this B12-photoCORM conjugate is demonstrated via synchrotron FTIR spectromicroscopy measurements on living cells. Intracellular photoinduced CO release prevents fibroblasts from dying under conditions of hypoxia and metabolic depletion, conditions that may occur in vivo during insufficient blood supply to oxygen-sensitive tissues such as the heart or brain.

We previously used synchrotron infrared microspectroscopy to describe the biochemical signature of skeletal muscle (biceps brachii) from the frozen ancient remains of a young man. In this current paper, we use light microscopy to assess the state of preservation of cellular components in the trapezius muscle from these same ancient remains and then use mid-infrared analysis at the Canadian Light Source synchrotron facility to further analyze the tissue. We compare spectra between the trapezius samples from the ancient remains and a recently deceased cadaver (control). Infrared spectra indicate preservation of secondary structure, with the α-helix being the principal component, along with triple helical portions of the protein backbone. Our mid-infrared analysis indicates an energy reserve in the skeletal muscle in the ancient remains.

Background: The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H(+) is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo., Methods: The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR., Results: We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO2 and corresponding decrease of HCO3(-) in the cell and in the surrounding medium., Conclusions: We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH., General Significance: Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

Femoral bone degeneration has been recognized as an important cause of lameness in broiler chickens for many years, but the pathogenesis of this condition has not been completely elucidated. The current work presents comprehensive analyses of changes associated with femoral bone degeneration based on findings from gross pathology, histopathology, biochemistry, and synchrotron-based imaging techniques. Gross lesions were predominantly seen in epiphysis and metaphysis of the proximal femur, and infrequently in distal femur, but we did not observe gross lesions in the diaphysis. Bone fractures were observed occasionally, but the most common lesions involved separation of articular cartilage of the femoral bone head, with progressive erosions of the subchondral bone. In advanced cases, on histopathological examination, changes in femoral bone were indicative of chondronecrosis and osteonecrosis. Computed tomography revealed that the degenerative process involves loss of trabecular bone. The course of the lesion development in the mineralized matrix appears to be coupled with increased bone resorption associated with excessive proliferation of pathologically altered osteoclasts. Light microscopy, Fourier transform infrared spectroscopy, and biochemical analysis provided consistent evidence that lowered protein content of the bone organic matrix is an integral component of femoral bone pathology, but these changes do not appear to be associated with excessive activity of matrix metalloproteinases. Taken together, our findings indicate that femoral bone degeneration is associated with structural changes occurring in both inorganic and organic matrix of the bone, but insufficiency in protein metabolism is most probably a primary aetiological factor in the natural history of femoral bone degeneration. However, it is important to stress that our findings do not negate the importance of bacterial infection in the evolution of this condition. Pathogens play a critical role in the progressive pathogenesis of this condition, which ultimately is manifested, in most instances, as femoral head necrosis.

The possibility of performing FT-IR spectromicroscopy experiments on individual living cells is the focus of considerable attention. Among the applications of interest, the obtainment of structural information in rapid measurements, with a time resolution of the minute or better, is a prized goal. In this work, we show that the use of synchrotron FT-IR spectromicroscopy allows one to extract weak spectral changes, of less than 10(-3) au per minute, in the absorption spectrum of single rod cells following photostimulation. We also show that absorption changes are accompanied by other optical effects due to changes in the real part of the refractive index of the cell. The use of two-dimensional correlation spectroscopy allows us to assign bands to specific molecular chromophores and to extract weak spectral variations in the presence of a noisy background.

Over the years Fourier-Transform Infrared (FTIR) spectroscopy has been widely employed in the structural and functional characterization of biomolecules. The introduction of infrared (IR) microscopes and of synchrotron light sources has created expectations that FTIR could become a generally viable technique to study both structure and reactivity in vivo, inside single cells, by performing measurements that up to a few years ago were the preserve of in vitro experiments on purified macromolecules. In this review we present the state-of-the-art in the application of FTIR spectromicroscopy as a technique for the study of structure and dynamics in single cells, we discuss the performance requirements for this application and review developments in sample handling methods.

We have tested an approach to identify putative cancer stem cells that involves measurement of the infrared absorption spectrum of individual cells in an aqueous environment, and their subsequent classification using multivariate data analysis techniques. Two primary esophageal cell lines were characterized: the immortalized normal esophageal epithelial cell line, Het-1A, and the esophageal adenocarcinoma cell line, OE33. In addition, we also evaluated spheroids, reflecting stem-like cell populations, which were derived from each parent cell line when grown in serum-free media. As differences in cell size appeared to be a strong discriminating factor, a correction needs to be performed to allow a reliable classification based on infrared absorption spectra. We demonstrated that stem-like cells derived from Het-1A could easily be discriminated on the basis of absorbance differences in the 1000-1200 cm(-1) spectral interval, whereas this was not possible for OE33. Furthermore, we found that changes due to aging of OE33 cells in culture dominated the infrared absorption spectra and somewhat limited the potential of this approach to identify stem-like cell populations using this in vitro model system.

We demonstrate the capability of synchrotron-based Fourier-Transform Infrared spectromicroscopy to detect metabolite formation in single, living cells of the unicellular algae Chlamydomonas reinhardtii. We show that the high brightness of the source provides a sufficient signal-to-noise ratio to detect small molecular species accumulating in a spot about 15 microm in size. Time resolved measurements are carried out on cells grown heterotrophically under low-light conditions to study the evolution of products of anaerobic metabolism. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

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.

We have recorded nanoscale topography and infrared chemical fingerprints of attomole layered lipids consisting of dimyristoylpho-sphatidylcholine on silicon and mica. Lipids deposited on mica built stacks consisting of up to 25 bilayers, each approximately 5 nm thick, spanning a range from 5-125 nm in height. Contrast evaluation as a function of layer thickness provides the near-field depth resolution.

beta-Defensins play an important role in both innate and adaptive immunity, displaying a direct anti-microbial activity against a wide variety of micro-organisms as well as interesting immuno-modulatory effects on host cells. Interaction with biological membranes appears to be a central theme in modulating these activities, leading to different consequences such as membrane lysis, translocation into the cytoplasm or transfer to a receptor. We have investigated the structuring of human beta-defensins (hBD2 and hBD3) and rationally designed variants, in relation to their interactions with real and model membranes. Biophysical methods, such as circular dichroism (CD), transmission or reflection IR and dye release were used to probe their structure/activity in the presence of model membranes, while fluorimetric and flow cytometric assays were used to investigate the effects on prokaryotic cells. Our results indicate that structural features, such as the helical N-terminal domains and oligomerisation at the membrane surface, may modulate the efficiency of membrane insertion and selectivity for microbial or host-cell membranes. We propose that both peptides interact with membranes as extended beta-sheet platforms that present amphipathic helices for insertion into the lipid bilayer.

The phase behavior of binary mixtures of egg sphingomyelin and cholesterol has been inspected by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in the amide I' band region of the spectrum. Because cholesterol does not have any major absorption bands in this region, effects seen in the spectra of mixtures of sphingomyelin and cholesterol can be attributed to the change in the lipid phase and to the interaction with cholesterol. It is shown that the temperature dependence of the overall bandwidth of the amide I' band displays a phase-specific behavior. In addition, it is observed that the amide I' band for a sample exhibiting phase coexistence can be described by a linear combination of the spectra of the individual lipid phases. Description of changes in the amide I' band shape and by that the study of possible hydrogen bonding interactions of sphingomyelin with cholesterol was assisted by the use of curve fitting. It turns out that the presence of hydrogen bonding between hydroxyl group of cholesterol and carbonyl group of sphingomyelin is obscured by the complexity of different possible hydrogen bonding and coupling between the N-H (N-D) and the CO group vibrations.

We used Fourier transform infrared spectromicroscopy in the attenuated total reflection configuration to study biochemical events associated with the response to light of an intact retina. We show that the technique is suitable for the detection in real time of molecular processes occurring in rod outer segments induced by light absorption. Two-dimensional correlation analysis was applied to the identification and interpretation of specific spectral changes associated to the evolution of the system. The technique allows us to observe an extensive protein translocation, which we interpret as arising from the release of transducin from the disk membrane and its redistribution from the outer segment towards the inner segment of rod cells. These results are in full agreement with our current understanding of retinal physiology and validate the technique as a useful tool for the study of complex molecular processes in intact tissue. [figure: see text] Spectral changes in the mid infrared region following exposure of an intact retina to light.

By using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and curve fitting we have examined temperature dependence and composition dependence of the shape of the carbonyl band in phosphatidylcholine/cholesterol model membranes. Membranes were hydrated either in excess water or in excess deuterated water. The studied binary mixtures exhibit different lipid phases at appropriate temperature and amount of cholesterol, among them also the so-called liquid-ordered phase. The results confirm that cholesterol has a significant indirect influence on the carbonyl band through conformational and hydration effects. This influence was interpreted in view of the known temperature composition phase diagrams for inspected binary mixtures. In addition, direct interaction was observed, which could point to the presence of hydrogen bond between cholesterol and carbonyl group. This direct interaction, though weak, might play at least a partial role in the stabilization of cholesterol-rich lipid domains in model and biological membranes.

Recent developments of element-specific microscopy techniques using synchrotron radiation are opening new opportunities for the analytical investigation of various heterogeneous materials. This article provides a general description of the operational principles of different microscopes allowing chemical and structural imaging combined with micro-spot spectroscopic analysis. Several selected examples are used to illustrate the potential of the synchrotron-based methods in terms of imaging and chemical sensitivity for identification of spatial variations in the composition of morphologically complex and nano-structured inorganic and organic materials, including biological samples.

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe(2+) but no terminal water/hydroxide bound to Fe(3+). The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Surface-enhanced resonance Raman scattering (SERRS) spectra were measured for the beta-carotene and lycopene carotenoids present in low-density lipoproteins (LDLs), which were isolated from human plasma and adsorbed on roughened silver surfaces. The silver surface was modified by formation of a self-assembled monolayer (SAM) of carboxylate-terminated linear alkanethiols in order to simulate the LDL binding region of the cellular LDL receptor. Thiols of different chain length were used to produce SAMs of varying thicknesses. It was shown that carotenoids are not released from the LDL particle upon adsorption onto the bare and thiol modified silver surfaces. The SERRS studies indicated that beta-carotene and lycopene were present in the shell of the LDL particle. The dependence of SERRS on the distance from the silver surface was different for beta-carotene and lycopene in LDL. This observation suggests that the two carotenoids are located in different places of the LDL particle.