X-ray microfluorescence (µXRF) imaging of Aspergillus nidulans cell wall mutants reveals biochemical changes due to gene deletions.

We used synchrotron X-ray fluorescence to create the first semiquantitative, submicron resolution, element distribution maps of P, S, K, and Ca, in situ, in fungal samples. Data collection was performed at the European Synchrotron Radiation Facility beam line ID21, Grenoble, France. We studied developing hyphae, septa, and conidiophores in Aspergillus nidulans, comparing wild type and two cell wall biosynthesis gene deletion strains. The latter encode sequential enzymes for biosynthesis of galactofuranose, a minor wall carbohydrate. Each gene deletion caused hyphal morphogenesis defects, and reduced both colony growth and sporulation 500-fold. Elemental imaging has helped elucidate biochemical changes in the phenotype induced by the gene deletions that were not apparent from morphological examination. Here, we examined S as a proxy for protein content, P for nucleic acid content, as well as Ca and K, which also have important metabolic roles. Element distributions in wild-type fungi reflect biological aspects already known or expected from other types of analysis; however, the application of X-ray fluorescence (XRF) imaging reveals aspects of gene deletion phenotypes that were not previously available. We have demonstrated that deleting a dispensable gene involved in galactose metabolism (ugeA) and one involved in biosynthesis of a minor cell wall component (ugmA) led to changes in hyphal elemental distribution that may have resulted from compromised wall composition.

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.

Restoration and spectral recovery of mid-infrared chemical images.

Fourier transform infrared (FTIR) microspectroscopy is a powerful technique for label-free chemical imaging that has supplied important chemical information about heterogeneous samples for many problems across a variety of disciplines. State-of-the-art synchrotron based infrared (IR) microspectrometers can yield high-resolution images, but are truly diffraction limited for only a small spectral range. Furthermore, a fundamental trade-off exists between the number of pixels, acquisition time and the signal-to-noise ratio, limiting the applicability of the technique. The recently commissioned infrared synchrotron beamline, infrared environmental imaging (IRENI), overcomes this trade off and delivers 4096-pixel diffraction limited IR images with high signal-to-noise ratio in under a minute. The spatial oversampling for all mid-IR wavelengths makes the IRENI data ideal for spatial image restoration techniques. Here, we measured and fitted wavelength-dependent point-spread-functions (PSFs) at IRENI for a 74× objective between the sample plane and detector. Noise-free wavelength-dependent theoretical PSFs are deconvoluted from images generated from narrow bandwidths (4 cm(-1)) over the entire mid-infrared range (4000-900 cm(-1)). The stack of restored images is used to reconstruct the spectra. Restored images of metallic test samples with features that are 2.5 µm and smaller are clearly improved in comparison to the raw data images for frequencies above 2000 cm(-1). Importantly, these spatial image restoration methods also work for samples with vibrational bands in the recorded mid-IR fingerprint region (900-1800 cm(-1)). Improved signal-to-noise spectra are reconstructed from the restored images as demonstrated for a mixture of spherical polystyrene beads in a polyurethane matrix. Finally, a freshly thawed retina tissue section is used to demonstrate the success of deconvolution achievable with a heterogeneous, irregularly shaped, biologically relevant sample with distinguishing spectroscopic features across the entire mid-IR spectral range.

Structure and composition of myelinated axons: a multimodal synchrotron spectro-microscopy study.

We report elemental mappings on the sub-cellular level of myelinated sciatic neurons isolated from wild type mice, with high spatial resolution. The distribution of P, S, Cl, Na, K, Fe, Mn, Cu was imaged in freeze-dried as well as cryo-preserved specimen, using the recently developed cryogenic sample environment at beamline ID21 at the European Synchrotron Radiation Facility (ESRF). In addition, synchrotron radiation based Fourier transform infrared (FTIR) spectromicroscopy was used as a chemically sensitive imaging method. Finally single fiber diffraction in highly focused hard X-ray beams, and soft X-ray microscopy and tomography in absorption contrast are demonstrated as novel techniques for the study of single nerve fibers.

Association among amyloid plaque, lipid, and creatine in hippocampus of TgCRND8 mouse model for Alzheimer disease.

Amyloid peptide (Aß) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30-50 µm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.

Dense-core and diffuse Abeta plaques in TgCRND8 mice studied with synchrotron FTIR microspectroscopy.

Plaques composed of the Abeta peptide are the main pathological feature of Alzheimer's disease. Dense-core plaques are fibrillar deposits of Abeta, showing all the classical properties of amyloid including beta-sheet secondary structure, while diffuse plaques are amorphous deposits. We studied both plaque types, using synchrotron infrared (IR) microspectroscopy, a technique that allows the chemical composition and average protein secondary structure to be investigated in situ. We examined plaques in hippocampal, cortical and caudal tissue from 5- to 21-month-old TgCRND8 mice, a transgenic model expressing doubly mutant amyloid precursor protein, and displaying impaired hippocampal function and robust pathology from an early age. Spectral analysis confirmed that the congophilic plaque cores were composed of protein in a beta-sheet conformation. The amide I maximum of plaque cores was at 1623 cm(-1), and unlike for in vitro Abeta fibrils, the high-frequency (1680-1690 cm(-1)) component attributed to antiparallel beta-sheet was not observed. A significant elevation in phospholipids was found around dense-core plaques in TgCRND8 mice ranging in age from 5 to 21 months. In contrast, diffuse plaques were not associated with IR detectable changes in protein secondary structure or relative concentrations of any other tissue components.

Synchrotron FTIR microspectroscopic analysis of the effects of anti-inflammatory therapeutics on wound healing in laminectomized rats.

Peridural scarring, or the excessive formation of scar tissue following spinal surgery, is one of the important contributing factors that result in persistent pain and disability in many individuals who have undergone elective back surgery. Treatment with anti-inflammatory agents following surgery may reduce oxidative stress and scarring, leading to a reduction in post-operative pain. We are using a surgical rat model to test the hypothesis that post-surgical inflammation and oxidative stress following laminectomy can be reduced by systemic administration of L: -2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin. OTC is a cysteine precursor required for the synthesis of glutathione, an important antioxidant. Quercetin is a flavonoid with anti-oxidant properties, found in fruits and vegetables. Synchrotron FTIR microspectroscopy data has been collected on OTC, quercetin and saline (control)-treated post-surgery animals, sacrificed at 3 and 21 days (n = 6 per age and treatment group). This paper presents preliminary IR results, supported by immunocytochemistry, on the heterogeneous distribution of biological components present in the healing tissue. The data collected on animals sacrificed at 3 and 21 days post-surgery will be combined in the future with data from animals sacrificed 63 days after surgery (representing a third time point) to evaluate the efficacy of the different treatments. Initial statistical analysis of ED1 immunohistochemistry results indicates a decrease in the number of activated macrophages 21 days post-surgery in the OTC-treated animals compared with the saline controls.

The creatine kinase/creatine connection to Alzheimer's disease: CK-inactivation, APP-CK complexes and focal creatine deposits.

Cytosolic brain-type creatine kinase (BB-CK), which is coexpressed with ubiquitous mitochondrial uMtCK, is significantly inactivated by oxidation, in Alzheimer's disease (AD) patients. Since CK has been shown to play a fundamental role in cellular energetics of the brain, any disturbance of this enzyme may exasperate the AD disease process. Mutations in amyloid precursor protein (APP) are associated with early onset AD and result in abnormal processing of APP, and accumulation of A beta peptide, the main constituent of amyloid plaques in AD brain. Recent data on a direct interaction between APP and the precursor of uMtCK support an emerging relationship between AD, cellular energy levels and mitochondrial function. In addition, recently discovered creatine (Cr) deposits in the brain of transgenic AD mice, as well as in the hippocampus from AD patients, indicate a direct link between perturbed energy state, Cr metabolism and AD. Here, we review the roles of Cr and Cr-related enzymes and consider the potential value of supplementation with Cr, a potent neuroprotective substance. As a hypothesis, we consider whether Cr, if given at an early time point of the disease, may prevent or delay the course of AD-related neurodegeneration.

Focally elevated creatine detected in amyloid precursor protein (APP) transgenic mice and Alzheimer disease brain tissue.

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.

Fourier transform infrared evaluation of microscopic scarring in the cardiomyopathic heart: effect of chronic AT1 suppression.

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.