Highlighting IR Spectrochemical Imaging of the Retina.

An emerging application of mid-IR spectrochemical imaging of the retina is its utility in studying the highly localized biomolecular alterations in the chemistry of retinal cell layers associated with several pathological conditions. Spatially resolved IR images highlight simultaneous chemical composition of the entire span of the retina in a label-free manner.

Extracting pure absorbance spectra in infrared microspectroscopy by modeling absorption bands as Fano resonances.

Midinfrared absorbance spectra obtained from spatially inhomogeneous and finite samples often contain scattering effects characterized by derivative-like bands with shifted peak positions. Such features may be interpreted and accurately modeled by Fano theory when the imaginary part of the complex dielectric function is small and Lorentzian in nature-as is the case for many biological media. Furthermore, by fitting Fano line shapes to isolated absorbance bands, recovery of the peak position and pure absorption strength can be obtained with high accuracy. Additionally, for small and optically soft spherical scatterers, recovery of one or the other of constant refractive index or radius (given approximate knowledge of the other) is possible.

Estimating and correcting interference fringes in infrared spectra in infrared hyperspectral imaging.

Short-term acclimation response of individual cells of Thalassiosira weissflogii was monitored by Synchrotron FTIR imaging over the span of 75 minutes. The cells, collected from batch cultures, were maintained in a constant flow of medium, at an irradiance of 120 µmol m-2 s-1 and at 20 °C. Multiple internal reflections due to the micro fluidic channel were modeled, and showed that fringes are additive sinusoids to the pure absorption of the other components of the system. Preprocessing of the hyperspectral cube (x, y, Abs(λ)) included removing spectral fringe using an EMSC approach. Principal component analysis of the time series of hyperspectral cubes showed macromolecular pool variations (carbohydrates, lipids and DNA/RNA) of less than 2% after fringe correction.

Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis.

Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm-1), proteins (1662 and 1608 cm-1) and fatty acids (2854, 2923, 2956 and 3012 cm-1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.

Thermal source Fourier transform infrared microtomography applied to Arctic sea ice diatoms.

We have used thermal source Fourier Transform Infrared (FTIR) microtomographic imaging to compare sea ice diatoms growing under different light conditions. A prototype tomography accessory was designed to have sufficient degrees of freedom to align any tilted cylindrical sample relative to the axis of rotation, minimizing the off-axis path traced during rotation. The lightweight device rests on the motorized stage to position the sample in the field-of-view and enable mosaic imaging. Reconstruction routines were tested with simulated and real phantoms, to assess limitations in the Radon back-projection method employed. The distribution and abundance of biochemicals is analysed for targets larger than a single FPA tile. Two and three dimensional (2D and 3D) FTIR spectrochemical images were obtained with a Focal Plane Array (FPA, nominal 1.1 µm pixel edges) for phantoms (polystyrene beads in polyvinyl alcohol matrix) and diatom cells harvested from land fast, first-year ice sites in Resolute Passage (74 43.628'N; 95 33.330'W) and Dease Strait (69° 1.11'N; 105° 21.29'W), Nunavut, Canada. The analysis of relative concentrations of organic matter within the encapsulating silica frustules of diatoms is important for a better understanding of both the physiological state and the individual cellular response to environmental pressures. Analysis of 3D FTIR images of Nitzschia frigida collected from beneath high (17-19 cm) and low (3-7 cm) snow depth revealed higher concentrations of lipids in diatoms collected under low snow cover, uniquely based on spectroscopically determined total 3D cell volume and biochemical content.

Multifunctional supramolecular polymer networks as next-generation consolidants for archaeological wood conservation.

The preservation of our cultural heritage is of great importance to future generations. Despite this, significant problems have arisen with the conservation of waterlogged wooden artifacts. Three major issues facing conservators are structural instability on drying, biological degradation, and chemical degradation on account of Fe(3+)-catalyzed production of sulfuric and oxalic acid in the waterlogged timbers. Currently, no conservation treatment exists that effectively addresses all three issues simultaneously. A new conservation treatment is reported here based on a supramolecular polymer network constructed from natural polymers with dynamic cross-linking formed by a combination of both host-guest complexation and a strong siderophore pendant from a polymer backbone. Consequently, the proposed consolidant has the ability to chelate and trap iron while enhancing structural stability. The incorporation of antibacterial moieties through a dynamic covalent linkage into the network provides the material with improved biological resistance. Exploiting an environmentally compatible natural material with completely reversible chemistries is a safer, greener alternative to current strategies and may extend the lifetime of many culturally relevant waterlogged artifacts around the world.

3D spectral imaging with synchrotron Fourier transform infrared spectro-microtomography.

We report Fourier transform infrared spectro-microtomography, a nondestructive three-dimensional imaging approach that reveals the distribution of distinctive chemical compositions throughout an intact biological or materials sample. The method combines mid-infrared absorption contrast with computed tomographic data acquisition and reconstruction to enhance chemical and morphological localization by determining a complete infrared spectrum for every voxel (millions of spectra determined per sample).

Dual band sensitivity enhancements of a VO(x) microbolometer array using a patterned gold black absorber.

Infrared-absorbing gold black has been selectively patterned onto the active surfaces of a vanadium-oxide-based infrared bolometer array. Patterning by metal lift-off relies on protection of the fragile gold black with an evaporated oxide, which preserves much of gold black's high absorptance. This patterned gold black also survives the dry-etch removal of the sacrificial polyimide used to fabricate the air-bridge bolometers. For our fabricated devices, infrared responsivity is improved 22% in the long-wave IR and 70% in the mid-wave IR by the gold black coating, with no significant change in detector noise, using a 300°C blackbody and 80 Hz chopping rate. The increase in the time constant caused by the additional mass of gold black is ∼15%.

Chemical structure and morphology of dorsal root ganglion neurons from naive and inflamed mice.

Fourier transform infrared spectromicroscopy provides label-free imaging to detect the spatial distribution of the characteristic functional groups in proteins, lipids, phosphates, and carbohydrates simultaneously in individual DRG neurons. We have identified ring-shaped distributions of lipid and/or carbohydrate enrichment in subpopulations of neurons which has never before been reported. These distributions are ring-shaped within the cytoplasm and are likely representative of the endoplasmic reticulum. The prevalence of chemical ring subtypes differs between large- and small-diameter neurons. Peripheral inflammation increased the relative lipid content specifically in small-diameter neurons, many of which are nociceptive. Because many small-diameter neurons express an ion channel involved in inflammatory pain, transient receptor potential ankyrin 1 (TRPA1), we asked whether this increase in lipid content occurs in TRPA1-deficient (knock-out) neurons. No statistically significant change in lipid content occurred in TRPA1-deficient neurons, indicating that the inflammation-mediated increase in lipid content is largely dependent on TRPA1. Because TRPA1 is known to mediate mechanical and cold sensitization that accompanies peripheral inflammation, our findings may have important implications for a potential role of lipids in inflammatory pain.

Correction: Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows.

Correction for 'Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows' by Ebrahim Aboualizadeh et al., Analyst, 2015, 140, 4046-4056.

Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows.

The chemical status of live sensory neurons is accessible with infrared microspectroscopy of appropriately prepared cells. In this paper, individual dorsal root ganglion (DRG) neurons have been prepared with two different protocols, and plated on glass cover slips, BaF2 and CaF2 substrates. The first protocol exposes the intact DRGs to 4 °C for between 20-30 minutes before dissociating individual neurons and plating 2 hours later. The second protocol maintains the neurons at 23 °C for the entire duration of the sample preparation. The visual appearance of the neurons is similar. The viability was assessed by means of trypan blue exclusion method to determine the viability of the neurons. The neurons prepared under the first protocol (cold exposure) and plated on BaF2 reveal a distinct chemical signature and chemical distribution that is different from the other sample preparations described in the paper. Importantly, results for other sample preparation methods, using various substrates and temperature protocols, when compared across the overlapping spectral bandwidth, present normal chemical distribution within the neurons. The unusual chemically specific spatial variation is dominated by a lack of protein and carbohydrates in the center of the neurons and signatures of unraveling DNA are detected. We suggest that cold shock leads to apoptosis of DRGs, followed by osmotic stress originating from ion gradients across the cell membrane leading to cell lysis.

Ultrasensitive chemical sensing through facile tuning defects and functional groups in reduced graphene oxide.

Herein, we report on a facile, low-cost, and efficient method to tune the structure and properties of chemically reduced graphene oxide (rGO) by applying a transient voltage across the rGO for ultrasensitive gas sensors. A large number of defects, including pits, are formed in the rGO upon the voltage activation. More interestingly, the number of epoxide and ether functional groups in the rGO increased after the voltage activation. The voltage-activated rGO was highly sensitive to NO2 with a sensitivity 500% higher than that of the original rGO. The lower detection limit can reach an unprecedented ultralow concentration of 50 ppb for NO2 sensing. Density functional theory (DFT) calculations revealed that the high sensitivity to NO2 is attributed to the efficient charge transfer from ether groups to NO2, which is the dominant sensing mechanism. This study points to a promising method to tune the properties of graphene-based materials through the creation of additional defects and functional groups for high-performance gas sensors.

High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron beams.

Conventional Fourier-transform infrared (FTIR) microspectroscopic systems are limited by an inevitable trade-off between spatial resolution, acquisition time, signal-to-noise ratio (SNR) and sample coverage. We present an FTIR imaging approach that substantially extends current capabilities by combining multiple synchrotron beams with wide-field detection. This advance allows truly diffraction-limited high-resolution imaging over the entire mid-infrared spectrum with high chemical sensitivity and fast acquisition speed while maintaining high-quality SNR.

Diagnosing malaria infected cells at the single cell level using focal plane array Fourier transform infrared imaging spectroscopy.

New methods are needed to rapidly identify malaria parasites in blood smears. The coupling of a Focal Plane Array (FPA) infrared microscope system to a synchrotron light source at IRENI enables rapid molecular imaging at high spatial resolution. The technique, in combination with hyper-spectral processing, enables imaging and diagnosis of early stage malaria parasites at the single cell level in a blood smear. The method relies on the detection of distinct lipid signatures associated with the different stages of the malaria parasite and utilises resonant Mie extended multiplicative scatter correction to pre-process the spectra followed by full bandwidth image deconvolution to resolve the single cells. This work demonstrates the potential of focal plane technology to diagnose single cells in a blood smear. Brighter laboratory based infrared sources, optical refinements and higher sensitive detectors will soon see the emergence of focal plane array imaging in the clinical environment.

High-spatial-resolution mapping of superhydrophobic cicada wing surface chemistry using infrared microspectroscopy and infrared imaging at two synchrotron beamlines.

The wings of some insects, such as cicadae, have been reported to possess a number of interesting and unusual qualities such as superhydrophobicity, anisotropic wetting and antibacterial properties. Here, the chemical composition of the wings of the Clanger cicada (Psaltoda claripennis) were characterized using infrared (IR) microspectroscopy. In addition, the data generated from two separate synchrotron IR facilities, the Australian Synchrotron Infrared Microspectroscopy beamline (AS-IRM) and the Synchrotron Radiation Center (SRC), University of Wisconsin-Madison, IRENI beamline, were analysed and compared. Characteristic peaks in the IR spectra of the wings were assigned primarily to aliphatic hydrocarbon and amide functionalities, which were considered to be an indication of the presence of waxy and proteinaceous components, respectively, in good agreement with the literature. Chemical distribution maps showed that, while the protein component was homogeneously distributed, a significant degree of heterogeneity was observed in the distribution of the waxy component, which may contribute to the self-cleaning and aerodynamic properties of the cicada wing. When comparing the data generated from the two beamlines, it was determined that the SRC IRENI beamline was capable of producing higher-spatial-resolution distribution images in a shorter time than was achievable at the AS-IRM beamline, but that spectral noise levels per pixel were considerably lower on the AS-IRM beamline, resulting in more favourable data where the detection of weak absorbances is required. The data generated by the two complementary synchrotron IR methods on the chemical composition of cicada wings will be immensely useful in understanding their unusual properties with a view to reproducing their characteristics in, for example, industry applications.

SR-FTIR imaging of the altered cadmium sulfide yellow paints in Henri Matisse's Le Bonheur de vivre (1905-6)--examination of visually distinct degradation regions.

SR-FTIR imaging has been used to map the mid-IR active photo-degradation phases in two thin sections of cadmium yellow paint removed from Henri Matisse's Le Bonheur de vivre (1905-1906, The Barnes Foundation). These samples represent both the darkened cadmium yellow foliage in the upper left of the work and the lightened cadmium yellow field beneath the central reclining figures. The altered cadmium yellow paints from both regions were found to contain cadmium carbonate (CdCO3), cadmium sulphate (CdSO4), and cadmium oxalate (CdC2O4). Each of these phases was imaged to determine their positions as a function of depth, with the aim of better understanding the role of each phase in the degradation mechanism. This speciation mapping is critical because cadmium oxalate was used in this period as an additive in cadmium yellow light. In addition, cadmium carbonate and cadmium sulphate were synthesis starting materials for cadmium yellow, and so their distribution throughout the paint layer can provide an indication of their roles. It was established that cadmium oxalate is localized at the surface of the paint layer, cadmium carbonate is found deeper in the layer but still enriched at the surface, and cadmium sulphate is distributed throughout the layer. This distribution, along with the chloride content of the paint suggesting a cadmium chloride starting material, is consistent with an alteration mechanism in which the cadmium sulphide is oxidized to sulphate and this is then converted to carbonate and oxalate. The relative solubilities of the three photo-degradation products are also relevant to their locations in the paint film.

Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy.

Understanding stem cell (SC) biology remains challenging and one of the few human tissues within which their in situ location is well characterized is the cornea. Individual human corneal epithelial cells were isolated from biopsies of live tissues using fluorescence-activated cell sorting (FACS); these were divided into putative SCs, transit-amplifying (TA) cells and terminally-differentiated (TD) cells. Employing synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy with a focal plane array (FPA), sub-cellular spatial resolution analysis of unstained isolated cells was achieved as a consequence of the brilliance of a 12 collimated beams arrangement allowing rapid spectral acquisition. Infrared (IR) spectra were extracted and pre-processed. Subsequent categorization with multivariate analysis of IR spectra derived from FPA images was used to investigate biomolecular changes between classes. A progressive segregation in cell-specific spectral categories with differentiation from SC to TA cell to TD cell was noted. Multiple different absorption peaks that discriminated putative SCs, TA cells and TD cells across DNA, protein and lipid spectral regions were identified. DNA regions (1080 and 1225 cm(-1)) and some protein regions (1443 cm(-1)) primarily segregated SCs from TA cells and TD cells, whilst amide regions and lipids (1,550, 1650 and 1740 cm(-1)) segregated TA cells and TD cells. Scanning electron microscopy images verified the external phenotypic characteristics of the different isolated cell types. These findings highlight the applicability of SR-FTIR microspectroscopy towards distinguishing SCs, TA cells and TD cells, and suggest that cellular classification via traditional methods of immunolabelling can be greatly aided by the use of spectral biomarkers.

Synchrotron FTIR reveals lipid around and within amyloid plaques in transgenic mice and Alzheimer's disease brain.

While the basis of neuronal degeneration in Alzheimer's disease (AD) continues to be debated, the amyloid cascade hypothesis remains central. Amyloid plaques are a required pathological marker for post mortem diagnosis, and Aß peptide is regarded by most as a critical trigger at the very least. We present spectrochemical image analysis of brain tissue sections obtained with the mid-infrared beamline IRENI (InfraRed ENvironmental Imaging, Synchrotron Radiation Center, U Wisconsin-Madison), where the pixel resolution of 0.54 × 0.54 µm(2) permits analysis at sub-cellular dimensions. Spectrochemical images of dense core plaque found in hippocampus and cortex sections of two transgenic mouse models of AD (TgCRND8 and 3×Tg) are compared with plaque images from a 91 year old apoE43 human AD case. Spectral analysis was done in conjunction with histochemical stains of serial sections. A lipid membrane-like spectral signature surrounded and infiltrated the dense core plaques in all cases. Remarkable compositional similarities in early stage plaques suggest similar routes to plaque formation, regardless of genetic predisposition or mammalian origin.

Toward optimal spatial and spectral quality in widefield infrared spectromicroscopy of IR labelled single cells.

Advancements in widefield infrared spectromicroscopy have recently been demonstrated following the commissioning of IRENI (InfraRed ENvironmental Imaging), a Fourier Transform infrared (FTIR) chemical imaging beamline at the Synchrotron Radiation Center. The present study demonstrates the effects of magnification, spatial oversampling, spectral pre-processing and deconvolution, focusing on the intracellular detection and distribution of an exogenous metal tris-carbonyl derivative 1 in a single MDA-MB-231 breast cancer cell. We demonstrate here that spatial oversampling for synchrotron-based infrared imaging is critical to obtain accurate diffraction-limited images at all wavelengths simultaneously. Resolution criteria and results from raw and deconvoluted images for two Schwarzschild objectives (36×, NA 0.5 and 74×, NA 0.65) are compared to each other and to prior reports for raster-scanned, confocal microscopes. The resolution of the imaging data can be improved by deconvolving the instrumental broadening that is determined with the measured PSFs, which is implemented with GPU programming architecture for fast hyperspectral processing. High definition, rapidly acquired, FTIR chemical images of respective spectral signatures of the cell 1 and shows that 1 is localized next to the phosphate- and Amide-rich regions, in agreement with previous infrared and luminescence studies. The infrared image contrast, localization and definition are improved after applying proven spectral pre-processing (principal component analysis based noise reduction and RMie scattering correction algorithms) to individual pixel spectra in the hyperspectral cube.

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques.

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.