Alignment maps of tissues: I. Microscopic elliptical polarimetry.

A method to image fiber alignment during mechanical testing of soft tissues was developed based on quantitative polarized light microscopy. Images were acquired after passing light through a rotating polarizer, a tissue sample, and an effective circular analyzer at multiple polarizer positions during uniaxial mechanical testing. The image set was analyzed offline using harmonic analysis to generate an alignment image, which contains the direction and strength of alignment at each image pixel. Alignment images of the entire tissue sample were generated every 3-5 s during the mechanical test allowing stress-strain behavior to be correlated with fiber alignment. Loading of fresh tissue-equivalent samples in the direction normal to the initial direction of fiber alignment revealed a spatially inhomogeneous realignment into the loading direction, with most realignment occurring near the free edges undergoing maximum lateral contraction and prior to significant load developing. Glutaraldehyde-fixed samples, in contrast, showed little realignment until yielding occurred.

Fiber alignment imaging during mechanical testing of soft tissues.

New advances in polarized light microscopy were used to image Congo red-stained cerebral amyloidosis in sharp relief. The rotating-polarizer method was used to separate the optical effects of transmission, linear birefringence, extinction, linear dichroism, and orientation of the electric dipole transition moments and to display them as false-color maps. These effects are typically convolved in an ordinary polarized light microscope. In this way, we show that the amyloid deposits in Alzheimer's disease plaques contain structurally disordered centers, providing clues to mechanisms of crystallization of amyloid in vivo. Comparisons are made with plaques from tissues of subjects having Down's syndrome and a prion disease. In plaques characteristic of each disease, the Congo red molecules are oriented radially. The optical orientation in amyloid deposited in blood vessels from subjects having cerebral amyloid angiopathy was 90° out of phase from that in the plaques, suggesting that the fibrils run tangentially with respect to the circumference of the blood vessels. Our result supports an early model in which Congo red molecules are aligned along the long fiber axis and is in contrast to the most recent binding models that are based on computation. This investigation illustrates that the latest methods for the optical analysis of heterogeneous substances are useful for in situ study of amyloid.

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Imaging linear birefringence and dichroism in cerebral amyloid pathologies

Alignment Maps of Tissues: II. Fast Harmonic Analysis for Imaging

Crystals grow in the mind’s eye by the addition of small units to a monolith each part of which is in fixed translational relation to every other part. Here, it is shown that growth can induce reversible twisting and untwisting of macroscopic crystals of hippuric acid (N-benzoylglycine, C9H9NO3) on the scale of radians. Crystals growing in undercooled melts of hippuric acid twist about the axis of elongation. At the same time the twisting is undone by new elastic stresses that build up as the crystal thickens. The dynamic interplay of twisting and untwisting ultimately fixes the crystal morphology. A correspondence between the optical properties of hippuric acid single crystals and twisted needles measured with a Mueller matrix microscope is established. The measured crystalloptical properties are in complete accord with the optical indicatrix rotating helically along the axis of elongation at the growth front, or counter-rotating so as to unwind earlier growth. The reversible morphological changes captur...

Reversible twisting during helical hippuric acid crystal growth.

Modulation techniques for measuring changes in optical birefringence, such as the rotating-polariser method (Wood & Glazer, 1980, J. Appl. Crystallogr. 13, 217), allow one to determine |sin δ|, δ = 2πLΔn/λ, Δn= double refraction, L = light path and λ = wavelength. However, they generally suffer from not providing absolute values of the optical retardance or are limited to relatively low retardance values. In addition, knowledge of the absolute phase is required when establishing the correct values of optical orientation information. In this paper, it is shown how the phase δ, and thus optical retardance, can be extracted from combining measurements of |sin δ| at different wavelengths. The new approach works on each single point of a 2-D picture without the need to correlate with neighbouring points. There is virtually no limit to the retardance, and the computational efforts are small compared with other methods (e.g. Ajovalasit et al. 1998, J. Strain Analysis 33, 75). When used with imaging techniques, such as the rotating polariser method of Glazer, Lewis & Kaminsky 1996 (Proc. R. Soc. London SeriesA452, 2751) this process has the potential to identify automatically optically anisotropic substances under the microscope. The algorithm derived in this paper is valid not only for birefringence studies, but can be applied to all studies of interfering light waves.

Lewis

Papers

Images of absolute retardance L.Deltan, using the rotating polariser method