Ira W. Levin

Infrared spectroscopic imaging for histopathologic recognition

The process of histopathology, comprising tissue staining and morphological pattern recognition, has remained largely unchanged for over 140 years. Although it is integral to clinical and research activities, histopathologic recognition remains a time-consuming, subjective process to which only limited statistical confidence can be assigned because of inherent operator variability. Although immunohistochemical approaches allow limited molecular detection, significant challenges remain in using them for quantitative, automated pathology. Vibrational spectroscopic approaches, by contrast, directly provide nonperturbing molecular descriptors, but a practical spectroscopic protocol for histopathology is lacking. Here we couple high-throughput Fourier transform infrared (FTIR) spectroscopic imaging of tissue microarrays with statistical pattern recognition of spectra indicative of endogenous molecular composition and demonstrate histopathologic characterization of prostatic tissue. This automated histologic segmentation is applied to routine archival tissue samples, incorporates well-defined tests of statistical significance and eliminates any requirement for dyes or molecular probes. Finally, we differentiate benign from malignant prostatic epithelium by spectroscopic analyses.

Raman spectra and vibrational assignments for dipalmitoyl phosphatidylcholine and structurally related molecules

Raman spectra of dipalmitoyl phosphatidylcholine and structurally related molecules are examined and vibrational transitions assigned for the C-C, phosphate and C-H stretching modes of these molecules. Particular emphasis is placed on the characteristics of the Raman spectra in the 2800-3000, 1000-1150 and 700-800 cm-minus 1 regions. It is found that hydrocarbon transitions dominate the spectra at the expense of those of the phosphate and choline groups. The methyl and methylene C-H stretching assignments have been clarified for the Raman spectra of phospholipid systems.

Imaging of collagen and proteoglycan in cartilage sections using Fourier transform infrared spectral imaging

OBJECTIVE To test the hypothesis that Fourier transform infrared (FTIR) spectral imaging, coupled with multivariate data processing techniques, can image the spatial distribution of matrix constituents in native and engineered cartilage samples. METHODS Tissue sections from native and trypsin-digested bovine nasal cartilage (BNC) and from engineered cartilage, generated by chick sternal chondrocytes grown in a hollow fiber bioreactor, were placed either on calcium fluoride windows for FTIR analysis or gelatinized microscope slides for histologic analysis. Based on the assumption that cartilage is predominantly chondroitin sulfate (CS) and type II collagen, chemical images were extracted from FTIR spectral imaging data sets using 2 multivariate methods: the Euclidean distance algorithm and a least-squares approach. RESULTS Least-squares analysis of the FTIR data of native BNC yielded a collagen content of 54 +/- 13% and a CS content of 37 +/- 16% (mean +/- SD). Euclidean distance analysis of measurements made on trypsin-digested BNC demonstrated only trace amounts of CS. For engineered cartilage, the CS content was significantly lower (15 +/- 5%), while the collagen content (73 +/- 6%) was significantly higher than biochemically determined values (CS 34%, collagen 5%, protein 61%). These differences are due to the fact that the dimethylmethylene blue assay overestimated the CS content of the tissue because it is not specific for CS, while the FTIR spectral imaging technique overestimated the collagen content because it lacks specificity for different proteins. CONCLUSION FTIR spectral imaging combines histology-like spatial localization with the quantitative capability of bulk chemical analysis. For molecules with a unique spectral signature, such as CS, the FTIR technique coupled with multivariate analysis can define a unique spatial distribution. However, for some applications, the lack of specificity of this technique for different types of proteins may be a limitation.

Infrared Spectroscopic Imaging: From Planetary to Cellular Systems

BY PINA COLARUSSO, LINDA H. KIDDER, IRA W. LEVIN, JAMES C. FRASER, JOHN F. ARENS, AND E. NEIL LEWIS* LABORATORY OF CHEMICAL PHYSICS, NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES, THE NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND 20892 (P.C., L.H.K., I.W.L., E.N.L.); VANGUARD RESEARCH, INC., FAIRFAX, VIRGINIA 22030 (J.C.F.); AND SPACE SCIENCES LABORATORY, UNIVERSITY OF CALIFORNIA, BERKELEY, CALIFORNIA 94720 (J.F.A.)

Vibrational spectra and carbon–hydrogen stretching mode assignments for a series of n‐alkyl carboxylic acids

The Raman and infrared spectra for the C4, C8, C12, and C16 carboxylic acid series were obtained at liquid nitrogen temperatures, in the 2900 cm−1 spectral region and were compared to spectra for selected branched and straight chain alkane systems. For hexadecanoic acid‐d3 the Fermi resonance interaction between the CD3 symmetric stretching mode and the overtone level of the CD3 asymmetric deformation vibration was examined as a function of physical state. In this example of a negative Fermi resonance perturbation, in which the lower frequency overtone component is more intense than the higher frequency fundamental, the coupling increases as the system passes from either the matrix isolated species or solution phase to the polycrystalline state. The spectra of the carboxylic acid series, as well as the spectra of 2,2,4‐trimethyl pentane and n‐octane, suggest that in the 2900 cm−1 region an analogous Fermi resonance coupling exists between the CH3 symmetric stretching vibration at ∼2938 cm−1 and the 2871 c...

Cholesterol-lipid interactions: An infrared and raman spectroscopic study of the carbonyl stretching mode region of 1,2-dipalmitoyl phosphatidylcholine bilayers

Abstract Infrared and Raman spectra were obtained for the 1690–1770 cm−1 carbonyl stretching mode region for 1,2-dipalmitoyl phosphatidylcholine (DPPC) bilayers in the anhydrous, partially hydrated and completely hydrated states. Spectral features at approx. 1740 and 1721 cm−1 are assigned to CO stretching modes associated with the 1- and 2-chain carbonyl groups, respectively. Splittings of the primary transitions at 1743, 1738, ∼1731 and ∼1721 cm−1 are attributed to rotational isomers involving the entire chain. Hydrogen bond formation between the fatty acid carbonyl and 3βOH cholesterol groups was investigated for anhydrous DPPC bilayers. Examination of frequencies, intensities and half-widths of the carbonyl bands indicates that no hydrogen bonding occurs at either of the two carbonyl sites. However, the addition of cholesterol to completely hydrated DPPC dispersions reduces the conformational inequivalence between the two fatty acid carbonyl groups by specifically perturbing the 2-chain. For cholesterol containing systems the carbonyl stretching mode transitions were also used to monitor lattice effects within the interface region as water binds to the bilayer head groups. Specifically, the addition of approx. 2 molecules of water per lipid molecule orders the lipid lattice and increases the bilayer packing density, while the subsequent addition of 4 molecules of water per lipid molecule releases the packing constraints within the interface region and thereby decreases the packing density.

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm−1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm−1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four (to ≈ 15°C) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.

Indium Antimonide (InSb) Focal Plane Array (FPA) Detection for Near-Infrared Imaging Microscopy

Near-infrared spectroscopy is a sensitive, noninvasive method for chemical analyses, and its integration with imaging technologies represents a potent tool for the study of a wide range of materials. In this communication the use of an indium antimonide (InSb) multichannel imaging detector for near-infrared absorption spectroscopic microscopy is described. In particular, a 128 × 128 pixel InSb staring array camera has been combined with a refractive optical microscope and an acousto-optic tunable filter (AOTF) to display chemically discriminative, spatially resolved, vibrational spectroscopic images of biological and polymeric systems. AOTFs are computer-controlled bandpass filters that provide high speed, random wavelength access, wide spectral coverage, and high spectral resolution. Although AOTFs inherently have a wide range of spectroscopic applications, we apply this technology to NIR absorption microscopy between 1 and 2.5 μm. The spectral interval is well matched to the optical characteristics of both the NIR refractive microscope and the AOTF, thereby providing near-diffraction-limited performance with a practical spatial resolution of 1 to 2 μm. Design principles of this novel instrumentation and representative applications of the technique are presented for various model systems.

High-Fidelity Fourier Transform Infrared Spectroscopic Imaging of Primate Brain Tissue

We demonstrate a new mid-infrared and near-infrared imaging approach which is ideally suited to microscopic applications. The method employs an indium antimonide (InSb) focal-plane array detector and a commercially available step-scan Fourier transform infrared spectrometer (FT-IR). With either a KBr or a CaF2 beamsplitter, images from 1 to 5.5 μm (10,000-1818 cm−1) can be rapidly acquired with the use of all the available pixels on the detector. The spectral resolution for each image is easily varied by changing the number of acquired images during the interferometer scan. We apply this technique to noninvasively generate image contrast in sections of monkey brain tissue and to relate these data to specific lipid and protein fractions. In addition, we describe several computational methods to highlight the spatial distributions of components within a sample.

High-Fidelity Raman Imaging Spectrometry: A Rapid Method Using an Acousto-optic Tunable Filter

In this communication, we describe a technique for obtaining high-fidelity Raman images and Raman spectra. The instrumentation provides the ability to rapidly collect large-format images with the number of image pixels limited only by the number of detector elements in the silicon charge-coupled device (CCD). Wavelength selection is achieved with an acousto-optic tunable filter (AOTF), which maintains image fidelity while providing spectral selectivity. Under computer control the AOTF is capable of µs tuning speeds within the operating range of the filter (400–1900 nm). The AOTF is integrated with the CCD and holographic Raman filters to comprise an entirely solid-state Raman imager containing no moving parts. In operation, the AOTF is placed in front of the CCD and tuned over the desired spectral interval. The two-dimensional CCD detector is employed as a true imaging camera, providing a full multichannel advantage over competitive Raman imaging techniques. Images and spectra are presented of a mixture of dipalmitoylphosphatidylcholine (DPPC) and L-asparagine, which serves as a model system for the study of both lipid/peptide and lipid/protein interactions in intact biological materials. The Raman images are collected in only several seconds and indicate the efficacy of this rapid technique for discriminating between multiple components in complex matrices. Additionally, high-quality Raman spectra of the spatially resolved microscopic regions are easily obtained.