David L. Wetzel

FT-IR Microspectroscopy Enhances Biological and Ecological Analysis of Algae

Abstract Fourier Transform Infrared (FT-IR) microspectroscopy provides an in situ, nondestructive chemical analysis of individual algal cells. Algae play key roles in nutrient cycling and energy flow through aquatic ecosystems and are pivotal in the sequestration of inorganic nutrients (e.g., carbon, nitrogen, and phosphorus) and transformation into organic forms. However, most methods used to measure algal nutritional and physiological changes are limited to detecting whole community responses because of the relatively large quantity of material needed for analysis (i.e., milligrams to grams). The spatial resolution achievable with infrared microspectroscopy allows for the analysis of macromolecular pools (e.g., proteins, lipids, carbohydrates) in individual cells that allows species specific measurements within heterogeneous microscopic communities. Initial applications characterized molecular pools within marine macroalgae and have since progressed toward ecologically based questions concerning algal physiological responses to changing nutrient availability in marine and freshwater ecosystems.

Chemical analysis of multiple sclerosis lesions by FT-IR microspectroscopy.

Fourier transform infrared microspectroscopy can be used to collect infrared spectra from microscopic regions of tissue sections. If spectra are collected along a grid pattern, then maps of chemical functional groups can be produced and correlated to tissue histopathology. In the present study, white matter from multiple sclerosis and control brains were examined. Mapping experiments were designed such that 17 spectra were collected at 200 microm intervals along a line that was partially or wholly within a multiple sclerosis lesion site or within a representative white matter region of control tissue. Data analysis was based on earlier in vitro studies which found that the carbonyl at 1740 cm(-1) increases when lipids become oxidized (Free Rad. Biol. Med. 16:591-601, 1994), and the amide I peak at approximately 1660 cm(-1) broadens when proteins become oxidized (FEBS Let. 362:165-170, 1995). The results indicated that the C=O to CH2 ratio (1740 cm(-1):1468 cm(-1)) was elevated at several collection points in lesion sites from multiple sclerosis brains compared to values from white matter of control brains. Inspection of the amide I peak at 1657 cm(-1) revealed that it was broadened towards 1652 cm(-1) in multiple sclerosis tissues but not control tissues. These results suggest that lipids and proteins could be oxidized at active multiple sclerosis lesion sites. The localization of these products to lesion sites supports a role for free radicals in the pathogenesis of multiple sclerosis.

High-performance liquid chromatographi determination of ferulic acid in wheat milling fractions as a measure of bran contamination☆

A high-performance liquid chromatographic method was developed for selective determination of ferulic acid in 7 min in the extracts from wheat flour and ground whole wheat at typical levels of 50 and 500 micrograms/g, respectively. Recovery of 99.9% was obtained when ferulic acid was extracted into dilute sulfuric acid, followed by enzymatic treatment of the extract with an alpha-amylase preparation. The chromatographic system included a 100-mm column packed with Hypersil 5 micron reversed-phase ODS operating isocratically with 12% methanol-citrate buffer (pH 5.4) mixture. The selectivity and sensitivity of both ultraviolet diode array and fluorescence detectors was investigated. The optimum wavelengths selected were 320 nm and 312 nm/418 nm respectively. Relative standard deviations of the analytical procedure were 2.43% and 5.10% for whole wheat and flour samples, respectively.

FT-IR spectroscopic imaging microscopy of wheat kernels using a Mercury–Cadmium–Telluride focal-plane array detector

Abstract A 64×64 Mercury–Cadmium–Telluride (MCT) focal-plane array detector attached to a Fourier transform infrared (FT-IR) microscope was used to spectroscopically image 8-μm-thick cross-sections of wheat kernels in the fingerprint region of the infrared spectrum. After fast-Fourier transformation of the raw image interferograms, the data can be displayed as either a series of spectroscopic images collected at individual wavelengths, or as a collection of IR spectra obtained at each pixel position in the image. Image contrast is achieved due to the intrinsic chemical nature of the sample at each pixel location in the image. Individual cell layers near the outer portion of the wheat kernel, as well as the primary root within the germ, can be clearly differentiated in the IR images as a result of this enhanced chemical contrast.

Fourier transform infrared (FT-IR) microspectroscopic census of single starch granules for octenyl succinate ester modification.

Fourier transform infrared (FT-IR) microspectroscopy was used to investigate reaction homogeneity of octenyl succinic anhydride modification on waxy maize starch and detect uniformity of blends of modified and native starches. For the first time, the level and uniformity of chemical substitution on individual starch granules were analyzed by FT-IR microspectroscopy. More than 100 starch granules of each sample were analyzed one by one by FT-IR microspectroscopy. In comparison to the native starch, modified starch had two additional bands at 1723 and 1563 cm(-1), indicative of ester formation in the modified starch. For the 3% modification level, the degree of substitution (DS) was low (0.019) and the distribution of the ester group was not uniform among starch granules. For the modified starch with DS of 0.073, 99% of individual starch granules had a large carbonyl band area, indicating that most granules were modified to a sufficient extent that the presence of their carbonyl ester classified them individually as being modified. However, the octenyl succinate concentration varied between granules, suggesting that the reaction was not uniform. When modified starch (DS = 0.073) was blended with native starch (3:7, w/w) to achieve a mixture with an average DS of 0.019, FT-IR microspectroscopy was able to detect heterogeneity of octenyl succinate in the blend and determine the ratio of the modified starch to the native starch granules.

Neuropathology of twitcher mice : examination by histochemistry, immunohistochemistry, lectin histochemistry and Fourier transform infrared microspectroscopy

The twitcher mouse is an authentic animal model of globoid cell leukodystrophy, which is a genetic disease that affects the lysosomal enzyme galactocerebroside β‐galactosidase. This enzyme deficiency causes one of its substrates, galactosylsphingosine (psychosine), to accumulate in myelin‐forming cells, which eventually results in their death. In the central nervous system, the death of oligodendrocytes is thought to cause a series of secondary pathological changes. In this study, several techniques were utilized to examine the neuropathology of two different brain regions in the twitcher mouse—the hindbrain and the cerebrum. Neuropathological changes were as follows: (1) demyelination was detected in the hindbrain but not in the cerebrum, (2) a high density of periodic acid‐Schiff‐positive cells were detected in the hindbrain and to a lesser extent in the cerebrum, (3) astrocyte gliosis was pronounced in both the hindbrain and cerebrum, and (4) macrophages were abundant in both the hindbrain and the cerebrum. We found that Periodic acid‐Schiff‐positive cells, astrocyte gliosis and macrophage infiltration were present in white and gray matter regions of the cerebrum, while they were generally absent from the granule and molecular layers of the cerebellum. In addition to these studies, we utilized the technique of Fourier transform infrared (FT‐IR) microspectroscopy to identify the in situ distribution of psychosine in the brains of twitcher mice. Evidence was obtained that indicates a large accumulation of psychosine in the hindbrain, and to a lesser extent in the white matter of the cerebrum in the twitcher mouse, but not the normal mouse. There was no evidence for the accumulation of psychosine in the molecular layer of the cerebellum from the twitcher or normal mouse. Our conclusions are as follows: (1) pathology is more advanced in the hindbrain compared to the cerebrum, which is likely due to the hindbrain becoming myelinated prior to the cerebrum, (2) demyelination is not necessary for the development of secondary pathological changes, (3) pathology is not limited to white matter in the cerebrum, (4) pathology is not present in all brain regions, i.e. the granule and molecular layers of the cerebellum are devoid of pathological changes, and (5) psychosine accumulates in both the cerebrum and hindbrain, but not in the molecular layer of the cerebellum in the twitcher mouse. This study demonstrates that FT‐IR microspectroscopy can be used to correlate chemical changes to histopathological changes in brains from twitcher mice, which suggests that FT‐IR microspectroscopy may be a useful tool for studies examining other brain diseases.

Physicochemical characterization of galactosyldiglycerides and their quantitation in wheat flour lipids by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method was developed for analyzing digalactosyldiglycerides (DGDG) and monogalactosyldiglyceride (MGDG) in polar lipids fractionated from lipid extracts of wheat or flour. Wheat lipid samples were prepared by solvent extraction, then fractionated on a silica gel packed open column. A Spherisorb ODS (octadecyl silane) column with methanol/water elution system was used for separation of glycolipids in the polar lipid fractions. The detection limit of the refractive index detector with interferometric optics was 0.25µg for both DGDG and MGDG. Separating on nonpolar bonded phase columns permitted us to differentiate, based on fatty acid composition and position, among components within the specific glycolipid classes. Semipreparative HPLC on analytical columns was used to subfractionate the polar lipids. The glycolipids were collected for functional group characterization. Approximately 35% of each DGDG subfraction was accounted for as carbohydrate. The absence of phosphorus precluded phospholipids. Fatty acid analysis by gas chromatography showed the first DGDG to be linoleic acid, whereas the second DGDG peak was composed of linoleic, oleic and palmitic acids. Mass spectrometric analysis of the first DGDG peak showed linoleic acid in both the SN-1 and 2 positions. Mass spectrometric analysis revealed that palmitic or oleic acid in the second peak was preferentially located on the SN-1 position; linoleic acid was on the SN-2 position.

Synchrotron Infrared Confocal Microspectroscopical Detection of Heterogeneity within Chemically Modified Single Starch Granules

This reports the first detection of chemical heterogeneity in octenyl succinic anhydride modified single starch granules using a Fourier transform infrared (FT-IR) microspectroscopical technique that combines diffraction-limited infrared microspectroscopy with a step size that is less than the mask projected spot size focused on the plane of the sample. The high spatial resolution was achieved with the combination of the application of a synchrotron infrared source and the confocal image plane masking system of the double-pass single-mask Continuμm® infrared microscope. Starch from grains such as corn and wheat exists in granules. The size of the granules depends on the plant producing the starch. Granules used in this study typically had a median size of 15 μm. In the production of modified starch, an acid anhydride typically is reacted with OH groups of the starch polymer. The resulting esterification adds the ester carbonyl (1723 cm−1) organic functional group to the polymer and the hydrocarbon chain of the ester contributes to the CH2 stretching vibration to enhance the intensity of the 2927 cm−1 band. Detection of the relative modifying population on a single granule was accomplished by ratioing the baseline adjusted peak area of the carbonyl functional group to that of a carbohydrate band. By stepping a confocally defined infrared beam as small as 5 μm × 5 μm across a starch granule 1 μm at a time in both the x and y directions, the heterogeneity is detected with the highest possible spatial resolution.

Microbeam molecular spectroscopy of biological materials

Summary Molecular vibrations of microscopic areas of biological tissue specimens are observed and measured to provide in situ , highly localized, chemical information. Fourier transform infrared (FT-IR) microspectroscopic interrogation of even single cells is done. Spectra at multiple points within single cells provide images for select wavelengths representing different chemical (functional group) distributions. Spatially resolved microspectroscopy allows study of heterogeneous materials and superimposition of chemical microstructure onto morphology. Recent ultra-spatial resolution has been achieved using a bright infrared source from a synchrotron (National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY) coupled with the Spectra-Tech, Inc. IRμs ™ scanning infrared microspectrometer. The combination of these powerful tools results in working at the diffraction limit with high signal/noise to obtain good spectra using 6×6 micrometer double aperturing and coadding only 16 scans. Individual adjacent cells can be readily interrogated one at a time to seek out chemical differences.

SINGLE FIBER ANALYSIS BY INTERNAL REFLECTION INFRARED MICROSPECTROSCOPY

Attenuated total reflection (ATR) is a convenient mode for single fiber analysis by infrared microspectroscopy, particularly when transmission spectra are difficult to obtain or when surface preferenced sampling is desirable. Textile finishes such as spin finishes, anti-static finishes, and permanent press finishes are revealed by ATR techniques. Bicomponent fibers may be analyzed by a combination of ATR techniques, transmission techniques, and spectral subtraction.