No abstract

Antibody binding to surface antigens in membranes is the primary event in the specific immune defence of vertebrates. Here we used force microscopy to study the dynamics of antibody recognition of mutant purple membranes from Halobacterium salinarum containing a genetically appended anti-Sendai recognition epitope. Ligation of individual anti-Sendai antibodies to their antigenic epitopes was observed over time. Their increase in number within a small selected area revealed an apparent kinetic on-rate. The membrane-bound antibodies showed many different conformations that ranged from globular to V-and Y-like shapes. The maximum distance of two Fab fragments of the same antibody was observed to be B18 nm, indicating an overall strong intrinsic flexibility of the antibody hinge region. Fab fragments of bound anti-Sendai antibodies were allocated to antigenic sites of the purple membrane, allowing the identification and localization of individual recognition epitopes on the surface of purple membranes.

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.