TYTELL, E.D.; Harvard University: Three-dimensional white muscle fiber orientations in zebrafish: An automatic technique for measuring fiber angles in fishes

The three-dimensional orientations of white muscle fibers in zebrafish, Brachydanio rerio, were measured using an automatic image analysis technique, based on confocal and two-photon microscopy. Previous anatomical studies have shown that white muscle fibers in fishes have a spiraling orientation down the body of the fish. The muscle is also divided into nested cones, called myomeres, which are separated by collagenous sheets called myosepta. These studies have assumed that many sequential fibers, separated by multiple myosepta, can be grouped in an overall trajectory that approximates a helix. However, it is unclear how this complex pattern is associated with body bending. Time-intensive manual dissection techniques previously used are not conducive to examining changes in fiber orientations with curvature, nor can they be easily applied to large comparative studies. Therefore, this study presents a technique for rapidly and automatically mapping three-dimensional fiber angles using autocorrelation-based image analysis. Whole zebrafish were embedded in plastic and sectioned optically using confocal or two-photon microscopy. Three-dimensional fiber angles were reconstructed from the axis of least curvature in the volumetric autocorrelation. The new technique confirms the previously observed spiral pattern. Additionally, to investigate the validity of the fiber trajectory concept � the idea that spiral patterns from different myomeres can be assembled into an overall helix � fiber angles on either side of a myoseptum were compared. Finally, to examine the relationship of curvature and fiber angles, zebrafish fixed with various degrees of curvature were also studied. Fiber orientations from bent animals show striking asymmetries between the left and right sides.