Mechanical properties of the arterial wall depend largely on orientation and density of collagen fiber bundles. Several methods have been developed for observation of collagen orientation and density; the most frequently applied collagen-specific manual approach is based on polarized light (PL). However, it is very time consuming and the results are operator dependent. We have proposed a new automated method for evaluation of collagen fiber direction from two-dimensional polarized light microscopy images (2D PLM). The algorithm has been verified against artificial images and validated against manual measurements. Finally the collagen content has been estimated. The proposed algorithm was capable of estimating orientation of some 35 k points in 15 min when applied to aortic tissue and over 500 k points in 35 min for Achilles tendon. The average angular disagreement between each operator and the algorithm was −9.3±8.6° and −3.8±8.6° in the case of aortic tissue and −1.6±6.4° and 2.6±7.8° for Achilles tendon. Estimated mean collagen content was 30.3±5.8% and 94.3±2.7% for aortic media and Achilles tendon, respectively. The proposed automated approach is operator independent and several orders faster than manual measurements and therefore has the potential to replace manual measurements of collagen orientation via PLM.

We have implemented and tested a new automatic method for the montage synthesis and three-dimensional (3D) reconstruction of large tissue volumes from confocal laser scanning microscopy data (CLSM). This method relies on maximization of the phase correlation between adjacent images. It was tested on a large specimen (a murine heart) that was cut into a number of individual sections with thickness appropriate for CLSM. The sections were scanned horizontally (in-plane) and vertically (perpendicular to the optical planes) to produce “tiles” of a 3D volume. Phase correlation maximization was applied to the montage synthesis of in-plane tiles and 3D alignment of optical slices within a given physical section. The performance of the new method is evaluated.

We have explored the possibility of measuring small changes of orientation within grains by electron backscattering diffraction (EBSD), in the scanning electron microscope. Conventional orientation maps (using EBSD) index the orientation of each position on the sample separately. This does not give accurate results for small differences of orientation. We have studied methods of measuring small changes in orientation by measuring the change from one EBSD pattern to the next directly, without indexing either. Previous workers have measured the change of position of a zone axis in the EBSD pattern. We have compared this with an alternative method, which we show to be superior, of measuring the shift of the peaks in the Hough transform from one diffraction pattern to the next. This means that we are measuring the change of orientation of sets of crystal planes within the grain, rather than measuring the change of orientation of zone axes. We show that it is possible, with a standard EBSD configuration, to measure the shift of the Kikuchi bands to a precision of about a 10th of a pixel, which corresponds to a change of orientation in the sample of about 0.1 mrad (0.006°).