Reflectance Microspectroscopy Research Articles

"Wax On, Wax Off": In Vivo Imaging of Plant Physiology and Disease with Fourier Transform Infrared Reflectance Microspectroscopy.

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well‐established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non‐destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change.

Revealing Inflammatory Indications Induced by Titanium Alloy Wear Debris in Periprosthetic Tissue by Label-Free Correlative High-Resolution Ion, Electron and Optical Microspectroscopy.

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.

Vis–NIR Reflectance Microspectroscopy of IDPs

Abstract Visible near-infrared (Vis–NIR) reflectance spectroscopy is a powerful nondestructive technique allowing the parent bodies identification of cosmomaterials such as meteorites, micrometeorites, and interplanetary dust particles (IDPs) studied in the laboratory. Previous studies showed that meteorites do not represent the full diversity of the solar system small bodies. We present here an experimental setup we developed for measuring Vis–NIR microspectroscopy of individual IDPs. We acquired diffuse Vis–NIR reflectance spectra of 15 particles ranging 7–31 μm in size. We discuss the requirements, the abilities, as well as the limitations of the technique. For sizes smaller than 17 μm, the slopes increase with decreasing particles sizes, while for sizes larger than 17 μm, the slopes are randomly distributed. The visible reflectance levels do not appear to be affected by the size of the IDPs, and show a bimodal distribution. Among the studied particles, we identified an IDP (L2079C18) exhibiting a feature at 0.66 μm, which is similar to the one observed by remote sensing on the surface of hydrated asteroids. This is the first detection of a hydration band in the reflectance spectrum of an IDP that could indicate a possible link between hydrated IDPs with hydrated asteroid surfaces.

Near-infrared optical reflectance microspectroscopy examining atrioventricular valvular rhythm of wildtype larval zebrafish as a pre-stage cardiac drug toxicity assay

Atrioventricular (AV) valve is a cardiac component of zebrafish that regulates the rhythm of blood flow, and may be utilized to assay unknown cardiac drug compounds. This research herein presents the construction of a near-infrared optical reflectance microspectroscopy (NIRORM), examining the rhythm of AV valve of wildtype zebrafish larvae in vivo as a preliminary drug toxicity assay, of which the experimental results indicate that NIRORM is effective in discriminating types of cardiac drugs based upon the stimulating or tranquilizing effects on AV valve, and such effects are highly dependent on the concentration of solution and duration of sample immersion.©2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Diffraction limited mid-infrared reflectance microspectroscopy with a supercontinuum laser.

Chemical mapping was demonstrated with a mid-infrared (MIR) microspectroscopy setup based on a supercontinuum source (SC) emitting in the spectral range from 1.55 to 4.5 µm and a MEMS-based Fabry-Pérot filter spectrometer. Diffraction limited spatial resolution in reflection geometry was achieved. A multilayer film consisting of different polymers and mixtures thereof was measured and results were compared to those gained with a conventional FTIR microscope equipped with a thermal MIR source. Results show that compared to thermal sources, the application of the SC source results in higher signal-to-noise ratios together with better spatial resolution and faster scanning. Furthermore, diffraction limited imaging of red blood cells was demonstrated for the first time in the MIR spectral region in reflection mode. The distinctive characteristics of the MIR spectral region in conjunction with the high brightness, spatial coherence and broadband nature of supercontinuum radiation show the potential for improving infrared microscopy significantly.

Probing hyperbolic polaritons using infrared attenuated total reflectance micro-spectroscopy

Abstract

Fourier transform mid-infrared-attenuated total reflectance (FTMIR-ATR) microspectroscopy for determining textural property of microwave baked tuber

Time series spectroscopic and textural analysis data were obtained from 5 varieties of tuber samples during microwave baking. These data were analyzed using evolutionary computing methods including partial least square discriminant analysis (PLSDA), partial least square regression (PLSR) and locally weighted partial least squares regression (LWPLSR). PLSDA was able to discriminate the tuber samples into three separate classes corresponding to their spectral properties. The predictability of spectra in full wavenumber region (4000–600 cm−1) and fingerprint region (1500–900 cm−1) were calculated using PLSR and LWPLSR and the relative performances of developed models were compared. It was observed that similar or even better predictions were obtained by models using spectra in the fingerprint region. Then, first-derivative and mean centering iteration algorithm (FMCIA) was carried out to select potential effective wavelengths and these selected wavelengths were further simplified using successive projections algorithm (SPA) for improving the model efficiency. Based on the FMCIA-SPA method for wavelength selection, the optimized models were established using LWPLSR for determination of tuber textural property (TTP) in terms of hardness, resilience, springiness, cohesiveness, gumminess and chewiness, with correlation coefficient of prediction (RP) of 0.797, 0.881, 0.584, 0.574, 0.728 and 0.690, respectively. The results of this study demonstrated that FTMIR-ATR spectroscopy could be used reliably and rapidly for the non-destructive assessment of textural property of microwave baked tuber.

Non-destructive provenance study of chert using infrared reflectance microspectroscopy

In this paper we explore the potential of non-destructive Infrared reflectance microspectroscopy (IRRMS) for the characterization of chert samples from 10 different geological outcrops in North America with emphasis on the Midcontinent. A principle objective of the project is to determine whether IRRMS analysis can provide a mineral-based signature for bedrock sources of chert artifacts. The technique utilizes a Bio-Rad Digilab FTS-40 Infrared spectroscope and microscope attachment that reflects an infrared beam off a chert specimen. Our findings indicate that spectral features from the molecules constituting minor and trace minerals in chert may provide sufficient characterization for chert sourcing.

Laser microperforated biodegradable microbial polyhydroxyalkanoate substrates for tissue repair strategies: an infrared microspectroscopy study

Flexible and biodegradable film substrates prepared by solvent casting from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) solutions in chloroform were microperforated by ultraviolet laser ablation and subsequently characterized using infrared (IR) microspectroscopy and imaging techniques and scanning electron microscopy (SEM). Both transmission synchrotron IR microspectroscopy and attenuated total reflectance microspectroscopy measurements demonstrate variations in the polymer at the ablated pore rims, including evidence for changes in chemical structure and crystallinity. SEM results on microperforated PHBHV substrates after cell culture demonstrated that the physical and chemical changes observed in the biomaterial did not hinder cell migration through the pores.

Mid-infrared reflectance microspectroscopy of human molars: Chemical comparison of the dentin–enamel junction with the adjacent tissues

Mid-infrared reflectance microspectroscopy was applied to the study of calcified tissues in human molars. Particular attention was focused on the dentin–enamel junction (DEJ). Major chemical components (apatite, organic matrix and water) and minor chemical components ( CO 3 2 - and HPO 4 2 - ions in apatite) were investigated. It was found that the average contents of organic matrix and CO 3 2 - ions increased in the order: enamel < DEJ < dentin. The smallest quantities of water and phosphates were observed in the DEJ, which finding can be explained by the loosest tissue packing in this location. The HPO 4 2 - ions were not detected in the DEJ, while in dentin their content was higher than in enamel. Differences between the reflection and transmission modes in the studies of calcified tissues were discussed.

Synchrotron infrared reflectance microspectroscopy study of film formation and breakdown on copper

This work demonstrates the utility of synchrotron infrared reflectance microspectroscopy in the far- and mid-IR for the determination of the composition of electrogenerated surface films formed during the general and localized corrosion of copper in alkaline and bicarbonate solutions. Back-reflection geometry has been employed to identify the anodic film formed on copper in 0.1 M NaOH solution at 0.3 V (versus a Ag/AgCl reference) to be mainly CuO. In 0.01 M NaHCO(3) solution general corrosion occurs with passive film formation below 0.2 V. The surface film at 0.2 V consisted mainly of bicarbonate, copper carbonate dihydroxide or malachite [CuCO(3).Cu(OH)(2)], Cu(OH)(2) and possibly some CuO. At higher potentials the passive film breaks down and localized corrosion occurs leading to the formation of pits. The composition of the surface films inside the pits formed at 0.6 V was found to be essentially the same as that outside but the relative amount of Cu(OH)(2) appears to be higher.

Reflectance Microspectroscopy of Natural Rock Samples in the Visible and near Infrared

We have collected reflectance spectra of various unprocessed rock samples in the 450-1,650 nm wavelength range with a spatial resolution of 60 and 120 microm (diameter) and using three illumination modes. Spectra taken in diffuse reflectance (dark field) mode are comparable to those obtained from macroscopic measurements and can provide the basis for mineral detection at that spatial scale. The spectral discrimination of the dark field mode is demonstrated to be consistent with the spatial resolution of the microscope for the samples examined. These results support the conclusion that reflectance microspectroscopy in the visible and near-infrared regions can be a valuable tool for understanding mineral formation at the spatial scale of tens of micrometers.

Attenuated total reflectance microspectroscopy mapping for the characterisation of bronze corrosion products

Samples collected from an archaeological object and an outdoor bronze monument as well as a bronze coupon naturally aged in an outdoor marine environment have been analysed in order to determine the composition of the corrosion and deposition compounds and map their stratigraphic distribution. Cross-sectioned samples and the bronze coupon have been placed onto the x-y-z motorized stage under the objective of an FTIR microscope and both punctual analysis and mapping of small areas was performed in ATR mode. The combination of visible light microscopy, which allows the microphotograph documentation of chemically heterogeneous samples, with FTIR-ATR mapping, is of the outmost importance in the characterisation of the corrosion features and behaviour of a wide variety of corroded bronze samples streamlining the selection of the most appropriate conservation materials and cleaning procedures to be adopted by conservators. Limitations, such as the spatial resolution of about 20 μm and silicon crystal opacity, are compared with the great advantage of obtaining distribution false colour plots of the different compounds. As non-destructive of the sample analytical technique ATR mapping can further be combined with other analytical techniques such as metallography, X-ray EDS and Raman spectroscopy. The limitation of the ATR technique in identifying important corrosion products, which do not absorb in the mid infrared, and the need to be combined with other type of analysis is also highlighted.

Characterisation of lubricants on ball bearings by FT‐IR using an integrating sphere

AbstractFourier transform‐infrared reflectance microspectroscopy has been used extensively for the examination of coatings on non‐planar surfaces such as ball bearings. While this technique offers considerable advantages, practical application has many drawbacks, some of which are easily overcome by the use of integrating sphere technology. This paper described the use of an integrating sphere for the quantification of thin layers of lubricant on the surface of ball bearings and the parameters that require optimisation in order to obtain reliable data. Several applications of the technique were discussed including determination of lubricant load on 12.7mm steel ball bearings and the examination of degraded lubricant on post‐mortem specimens. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Fourier Transform Infrared Reflectance Microspectroscopy Study of Bacillus Subtilis Engineered without Dipicolinic Acid: The Contribution of Calcium Dipicolinate to the Mid-Infrared Absorbance of Bacillus Subtilis Endospores

Mid-infrared spectra of spores of two strains of Bacillus subtilis, PS832 (wild-type) and FB122 (sleB spoVF), that are isogenic except for the two mutations in FB122 were obtained by Fourier transform infrared (FT-IR) reflectance microspectroscopy. The mutations in FB122 cause the spores of this strain to be devoid of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a biomarker characteristic of bacterial spores. Analysis of these two strains by difference spectroscopy revealed a spectrum similar to that of calcium dipicolinate (CaDPA), a chelate salt of DPA. This difference spectrum was compared to mid-infrared spectra of both DPA and CaDPA, and was attributed to CaDPA only. This is the first report known to the authors of a genetically engineered organism being used to identify the spectral contribution of a particular cellular component.

Fourier Transform Infrared Microspectroscopy of Plant Tissues

Fourier transform infrared (FT-IR) microspectroscopy was applied to three distinct types of plant tissue. Reflectance microspectroscopy of nutshells highlighted the differences between the chemistries of the inner and outer surfaces and the tissue as a whole. The outer surfaces were suberized, while the inner surfaces contained absorbances indicative of lignin or tannins or both. Transmission microspectroscopy was used to follow the changes in cell wall structure and composition of flax epidermal cells during development and showed that initial development was accompanied by suberin and lignin deposition, which was followed by polysaccharide deposition characteristic of secondary cell wall formation. These results were compared with those obtained from bamboo. Transmission microspectroscopy was also used to study the infection of potato tubers by Erwinia carotovora ssp. carotovora in aerobic and anaerobic conditions. The spectra suggested that both infective conditions produced cell wall degradation, whereas anaerobic infection was accompanied by extensive breakdown of starch and plasma membrane.

ETHANOL INDUCES HETEROGENEOUS REDUCTION OF CYTOCHROME aa3 WITHIN PERFUSED RAT LIVER LOBULE ASSESSED USING MICROSPECTROSCOPY

The redox state of cytochrome aa3 was measured at microspots (20 microns diameter) within the lobule of perfused rat livers, using reflectance microspectroscopy, and the effect of ethanol infusion on sublobular distribution of the redox states was evaluated. A sigmoidal relationship was observed between oxygen delivery and the reduction of cytochrome aa3 in both the periportal and pericentral regions of the liver lobule when the influent O2 concentration was decreased in a graduated manner. This sigmoidal curve was shifted to the more reduced state by ethanol infusion, with ethanol (25-100 mM) increasing the degree of cytochrome aa3 reduction in a dose-dependent manner according to the distance from the periportal region along a sinusoid. This increase was spatially heterogeneous within a liver lobule. These data indicate that ethanol accelerates cytochrome aa3 reduction, with a distinct gradient of reduction along sinusoids but a heterogeneous distribution within the liver lobule.

Characterization and Application of a Grazing Angle Objective for Quantitative Infrared Reflection Microspectroscopy

A grazing angle objective on an infrared microspectrometer is studied for quantitative spectroscopy by considering the angular dependence of the incident intensity within the objective's angular aperture. The assumption that there is no angular dependence is tested by comparing the experimental reflectance of Si and KBr surfaces with the reflectance calculated by integrating the Fresnel reflection coefficient over the angular aperture under this assumption. Good agreement was found, indicating that the specular reflectance of surfaces can straightforwardly be quantitatively integrated over the angular aperture without considering nonuniform incident intensity. This quantitative approach is applied to the thickness determination of dipcoated Krytox on gold. The infrared optical constants of both materials are known, allowing the integration to be carried out. The thickness obtained is in fair agreement with the value determined by ellipsometry in the visible. Therefore, this paper illustrates a method for more quantitative use of a grazing angle objective for infrared reflectance microspectroscopy.

Infra‐red microspectroscopy as a complementary technique to electron‐probe microanalysis for the investigation of natural corrosion on potash glasses

SummaryNatural corrosion crusts on potash—lime—silica glasses, that have been buried in soil for very long periods, were examined using infra‐red reflectance microspectroscopy and electron‐probe techniques. Individually these techniques are useful: infra‐red provides information on which functional groups are present, but is not good at distinguishing between cations; electron‐probe microanalysis reveals the composition of a sample, but cannot explain how the elements are combined. By using both techniques on the same sample areas the corrosion was shown to be composed of layers of amorphous silica gel with precipitates such as carbonate‐substituted calcium phosphate, calcium sulphate and manganese dioxide.

Measurement of redox states of mitochondrial cytochrome aa3 in regions of liver lobule by reflectance microspectroscopy.

To evaluate the sublobular distribution of oxygen tension, the redox states of mitochondrial cytochrome aa3 were assessed in local regions of the liver lobule by reflectance microspectroscopy. The reflected light from two focused microspots (20 microns in diam) on the surface of the perfused liver, placed on the stage of microscope, was conducted to the spectrophotometer with two separate light guides, giving the reflectance spectra. The redox state of cytochrome aa3 was calculated from the difference in reflectance between 603 and 630 nm. The degree of cytochrome reduction increased with the stepwise decline of the influent or effluent oxygen concentrations. The local oxygen concentration in periportal region for the half-maximal reduction of cytochrome aa3 was higher than that in pericentral region. In addition, the degree of cytochrome aa3 reduction increased with the distance from the periportal region, reflecting a physiological gradient of oxygen tension along the sinusoidal flow, although with an intersinusoidal heterogeneity within liver lobule.