Search

The binding characteristics and inhibition kinetics of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium with imidazolyl carboxylic acids were examined. Findings indicated that these compounds are the most potent inhibitors of P-450 BM3. Data showed evidence for interdomain communication between the heme diflavin domains of the flavocytochrome. Binding of the inhibitors to the P-450 BM3 active site resulted from the binding of either pyridine nucleotide or fatty acid substrates at key catalytic residues.

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.)