Three different Raman microspectroscopic imaging methodologies using a single experimental configuration are compared; namely, point and line mapping, as representatives of serial imaging approaches, and direct or wide-field Raman imaging employing liquid-crystalline tunable filters are surveyed. Raman imaging data acquired with equivalent low-power 514.5-nm laser excitation and a cooled CCD camera are analyzed with respect to acquisition times, image quality, spatial resolution, intensity profiles along spatial coordinates, and spectral signal-to-noise ratios (SNRs). Point and line mapping techniques provide similar SNRs and reconstructed Raman images at spatial resolutions of approximately 1.1 microm. In contrast, higher spatial resolution is obtained by direct, global imaging (approximately 313 nm), allowing subtle morphological features on test samples to be resolved.
We characterize a visible reflectance hyperspectral imaging system for noninvasive, in vivo, quantitative analysis of human tissue in a clinical environment. The subject area is illuminated with a quartz-tungsten-halogen light source, and the reflected light is spectrally discriminated by a liquid crystal tunable filter (LCTF) and imaged onto a silicon charge-coupled device detector. The LCTF is continuously tunable within its useful visible spectral range (525-725 nm) with an average spectral full width at half-height bandwidth of 0.38 nm and an average transmittance of 10.0%. A standard resolution target placed 5.5 ft from the system results in a field of view with a 17-cm diameter and an optimal spatial resolution of 0.45 mm. The measured reflectance spectra are quantified in terms of apparent absorbance and formatted as a hyperspectral image cube. As a clinical example, we examine a model of vascular dysfunction involving both ischemia and reactive hyperemia during tissue reperfusion. In this model, spectral images, based upon oxyhemoglobin and deoxyhemoblobin signals in the 525-645-nm region, are deconvoluted using a multivariate least-squares regression analysis to visualize the spatial distribution of the percentages of oxyhemoglobin and deoxyhemoglobin in specific skin tissue areas.
The discovery of beta-sheet structure in Alzheimer's amyloid fibrils, and then in many other disease-related protein fibrils, has led to the widely believed view that beta-sheet formation is the general mechanism of aberrant protein aggregation leading to disease. This notion is further reinforced by recent findings, which indicate that normal proteins can be induced to form beta-sheet fibrils in vitro. Alzheimer's disease, a paradigm proteopathy, is accompanied by the formation of two distinct aggregates, amyloid fibrils and paired helical filaments (PHFs). Electron microscope images of PHFs show pairs of twisted ribbons with 80 nm periodicity. However, there is little information of the molecular structure of PHFs, as previous studies have failed to identify signs of regular structure. Using far-UV circular dichroism and Fourier-transformed infrared spectroscopy, we find that PHFs are comprised of alpha-helices. This is remarkable as tau-protein, PHF's primary constituent, has a high abundance of helix-breaking amino acids and is unstructured in solution. We also find that PHFs are very stable, as judged by their high melting temperature and resistance to protease digestion. PHFs are the first example of pathological aggregation associated to the formation of alpha-helix.
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