The beamline design, microscope specifications, and initial results from the new mid-infrared beamline (IRENI) are reviewed. Synchrotron-based spectrochemical imaging, as recently implemented at the Synchrotron Radiation Center in Stoughton, Wisconsin, demonstrates the new capability to achieve diffraction limited chemical imaging across the entire mid-infrared region, simultaneously, with high signal-to-noise ratio. IRENI extracts a large swath of radiation (320 hor. × 25 vert. mrads ² ) to homogeneously illuminate a commercial infrared (IR) microscope equipped with an IR focal plane array (FPA) detector. Wide-field images are collected, in contrast to single-pixel imaging from the confocal geometry with raster scanning, commonly used at most synchrotron beamlines. IRENI rapidly generates high quality, high spatial resolution data. The relevant advantages (spatial oversampling, speed, sensitivity, and signal-to-noise ratio) are discussed in detail and demonstrated with examples from a variety of disciplines, including formalin-fixed and flash-frozen tissue samples, live cells, fixed cells, paint cross-sections, polymer fibers, and novel nanomaterials. The impact of Mie scattering corrections on this high quality data is shown, and first results with a grazing angle objective are presented, along with future enhancements and plans for implementation of similar, small-scale instruments.

Infrared (IR) spectroscopy has been used for decades to study collagen in mammalian tissues. While many changes in the spectral profiles appear under polarized IR light, the absorption bands are naturally broad because of tissue heterogeneity. A better understanding of the spectra of ordered collagen will aid in the evaluation of disorder in damaged collagen and in scar tissue. To that end, collagen spectra have been acquired with polarized far-field (FF) Fourier Transform Infrared (FTIR) imaging with a Focal Plane Array detector, with the relatively new method of FF optical photothermal IR (O-PTIR), and with nano-FTIR spectroscopy based on scattering-type scanning near-field optical microscopy (s-SNOM). The FF methods were applied to sections of intact tendon with fibers aligned parallel and perpendicular to the polarized light. The O-PTIR and nano-FTIR methods were applied to individual fibrils of 100–500 nm diameter, yielding the first confirmatory and complementary results on a biopolymer. We observed that the Amide I and II bands from the fibrils were narrower than those from the intact tendon, and that both relative intensities and band shapes were altered. These spectra represent reliable profiles for normal collagen type I fibrils of this dimension, under polarized IR light, and can serve as a benchmark for the study of collagenous tissues.

A recent upgrade to the optics configuration of a thermal source FTIR microscope equipped with a focal plane array detector has enabled rapid acquisition of high magnification spectrochemical images, in transmission, with an effective geometric pixel size of ∼1 × 1 μm² at the sample plane. Examples, including standard imaging targets for scale and accuracy, as well as biomedical tissues and microorganisms, have been imaged with the new system and contrasted with data acquired at normal magnification and with a high magnification multi-beam synchrotron instrument. With this optics upgrade, one can now conduct rapid biodiagnostic ex vivo tissue imaging in-house, with images collected over larger areas, in less time (minutes) and with comparable quality and resolution to the best synchrotron source FTIR imaging capabilities.

Amyloid peptide (Aβ) 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.

In this study, we demonstrate the first in situ detection of usnic acid (UA) in selected species of the lichen Cladonia, using FPA-FTIR imaging and Raman microscopy. Fruticose lichens present a variety of defensive mechanisms, one of which is the production of UA. This polyketide secondary metabolite, produced by certain lichenized fungi, has a protective function for the lichen that includes a strong absorption in the ultraviolet range. Upon confirming the distinct spectral signature of UA in lichen tissue, we mapped its distribution in Cladonia arbuscula, Cladonia uncialis and Cladonia sulphurina tissues. Spectroscopic images were obtained from cryosectioned lichen fragments embedded in media and from hand-sectioned fragments that were media-free. UA was present in the pycnidia, and younger walls of C. arbuscula and C. uncialis, the spore-producing region of a C. uncialis apothecium, and in both the younger and older soredia of C. sulphurina. The localization of UA in lichens is an important precursor to future work that includes the identification of the gene cluster responsible for its biosynthesis. Our results show that FTIR and Raman imaging can be an effective way to study the distribution of natural products in lichens with micron-scale precision.

The creatine (Cr) energy system has been implicated in Alzheimer’s disease (AD), including reductions in brain phosphoCr and Cr kinase, yet no studies have examined the neurobehavioral effects of Cr supplementation in AD, including the 3xTg mouse model. This studied investigated the effects of Cr supplementation on spatial cognition, plasticity- and disease-related protein levels, and mitochondrial function in the 3xTg hippocampus. Here, 3xTg mice were fed a control or Cr-supplemented (3% Cr (w/w)) diet for 8–9 weeks and tested in the Morris water maze. Mitochondrial oxygen consumption (Seahorse) and protein levels (Western blots) were measured in the hippocampus in subsets of mice. Overall, 3xTg females exhibited impaired memory as compared to males. In females, Cr supplementation decreased escape latency and was associated with increased spatial search strategy use. In males, Cr supplementation decreased the use of spatial search strategies. Pilot data indicated mitochondrial enhancements with Cr supplementation in both sexes. In females, Cr supplementation increased CREB phosphorylation and levels of IκB (NF-κB suppressor), CaMKII, PSD-95, and high-molecular-weight amyloid β (Aβ) species, whereas Aβ trimers were reduced. These data suggest a beneficial preventative effect of Cr supplementation in females and warrant caution against Cr supplementation in males in the AD-like brain.