Non Radiative Excitation Fluorescence Microscopy: A New Method for Studying Membrane Adhesion at the Nanoscale

Abstract

Amyloid fibrils are associated with many diseases; Amyloid-b in particular is related to Alzheimer's Disease. We present the structure of an Ab(1-42) fibril determined by cryo-EM to a resolution of 4.0 Å . The fibril consists of two intertwined protofilaments in which individual subunits form a 'LS'-shaped topology revealing a previously unknown dimer interface, different from previous solid-state NMR models. All 42 aminoacids are well resolved in the EM density map, including the N-terminal part. The high resolution structure is in agreement with solid-state NMR and X-ray diffraction experiments. 824-Pos Board B594Microtubules are dynamic, filamentous structures that are essential for many different cellular processes. These processes often rely on a number of microtubule associated proteins (MAPs) that either regulate or carry out these functions. Characterizing interactions between MAPs and microtubules is crucial to understanding of how these processes are carried out. Recent advances in cryoelectron microscopy have made observing interactions between microtubules and several different MAPs at near atomic resolution possible. However, the resolution of these structures is variable, depending on the MAP. Recent studies have shown that the microtubule lattice is flexible and different stabilizing drugs and MAPs affects this flexibility. These results suggest that distortions in the microtubule lattice are limiting overall resolution. Rather than aligning whole segments of microtubules (like in conventional microtubule reconstructions), we have developed a technique that aligns patches of tubulin. By subtracting the surrounding microtubule density and leaving a patch of tubulin dimers, each patch can be aligned independently from the bulk of the microtubule. We have tested this 'patch refinement' technique on a synthetically, distorted dataset. Using traditional reconstruction techniques yielded a low resolution structure, but upon application of the patch refinement technique, the resulting structure was nearly indistinguishable from an equivalent undistorted, synthetic dataset. The patch refinement method has also been applied to an experimental dataset that was resolved to >5Å using traditional methods. Following patch refinement, the resolution increased to <3.6Å . Using the alignment parameters following patch refinement, we were able to trace the path of individual protofilaments and observe distortions in the lattice. Further use and development of this technique will allow us to analyze microtubule distortions and work towards obtaining an atomic structure of a microtubule.

Supercritical Angle Fluorescence Microscopy and Spectroscopy

“…In either case, the sensitivity of this label-free fluorescent SAF assay will be limited by the fluorescence signal available after prolonged use of the capillary sensor and by the time course of photobleaching of the immobilized dye. Conceptually, the technique is reminiscent of nanometerscale axial resolution with nonradiative excitation (64).…”