3D Nanometer Tracking of Motile Microtubules on Reflective Surfaces

Biomolecular motor-driven nanodevices are dynamic, soft systems exhibiting rapid energy flow and mechanical motion. To understand the spatial arrangement between nano-objects inthesedevices,afast,non-destructiveimagingtechniquewith nanometer spatial resolution in 3D is required. We employ fluorescence interference contrast microscopy (FLIC) to image the interaction between kinesin-driven microtubules (MTs) and nanoscopic surface structures. We directly image the geometry of crossing MTs with high temporal resolution and investigate how the leading tips of motile MTs explore their environment. We show directly that the length of the free MT tip, which is defined by the average distance between motors on the surface, determines the ability to overcome obstacles. Moreover, we demonstrate that FLIC microscopy in combination with fluorescently-labeled, motile MTs is a versatile tool to scan the geometry of engineered surfaces with nanometer height precision. This method, which is compatible with any kind of reflecting surface, permits dynamic and precise data acquisition in a highly parallel manner using a standard epi-fluorescence microscope. Active movement is a key ability of biological nanomachines, and finds applications in a number of hybrid bionanodevices. [1‐3] Molecular shuttles driven by motors and carrying cargo have been designed by us [4‐6] and others [7‐12] to address the need for nanoscale transport, positioning, and assembly systems. Our approach utilizes MTs—hollow, cylindrical, proteinaceous filaments that can be formed in vitro by self-assembly of tubulin heterodimers—as shuttles (Figure 1). MTs have an outer diameter of 25nm and can be as long as several micrometers. In a typical gliding motility assay, fluorescently-labeled MTs are transported at speeds of about 1 m ms � 1 by kinesin-1 motor proteins immobilized on a substrate surface. Kinesin motors—dimeric proteins capable of generating several piconewtons of mechanical force from the hydrolysis of adenosine 5 0 -triphosphate (ATP)—step towards the so-called ‘‘plus’’ end of the MTs. [13] Typically, several dozen motors contribute to the forward movement of one MT. Due to the high stiffness of MTs, the path that an undisturbed MT follows is normally rather straight (trajectory persistence length � 0.1 mm [14] ) and we expect a constant elevation above the surface. [15] As cargo, a wide range of objects [16‐18] including microspheres, [5] quantum dots, [19]