The fundamental goal of microscopic imaging is to visualize and identify small objects and observe their motion. Of the many available techniques, single particle tracking has been an immensely powerful tool in the life sciences for studying the motion of individual objects and thereby the function of a large variety biological processes. Despite considerable advances, following the motion of individual molecules on the relevant time and size scale has remained an insurmountable challenge. Here, we propose to construct a novel optical microscope based on interferometric scattering (iSCAT) detection that will bridge the considerable gap between the temporal and spatial accuracy provided by current single molecule methods.
The primary objective of this work is to utilize the unique imaging properties of interferometric scattering detection to directly visualize and thereby understand the power stroke of Myosin V. Myosin V is a molecular motor, a cargo transporter that moves along actin filaments while hydrolyzing ATP. A wealth of knowledge concerning myosin’s function has been acquired over the past decades. Nevertheless, many of the fundamental questions concerning the mechanism of the power stroke for these motors remain, largely due to fundamental limitations associated with the employed techniques. In this respect, the application of high-speed iSCAT to study the motion of Myosin V will represent a significant step forward in our understanding of the functionality of this important motor.
We aim that this work will establish iSCAT as a viable and powerful in vitro imaging platform with capabilities that are several orders of magnitude beyond what is currently possible with state of-the-art single particle tracking approaches. As a consequence, this proposal will include specifics with respect both the biological and the technical challenges involved with the proposed studies.
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