Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy

Abstract

Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.

Direct observation of individual tubulin dimers binding to growing microtubules

“…1B). MTs were detected label-free, as expected (38)(39)(40), and tubulinbound gold nanoparticles (tubulin-gold) appeared as points of high contrast (Fig. 1C).…”

Single-Nanoparticle Tracking with Angstrom Localization Precision and Microsecond Time Resolution

“…Recently, interferometric scattering microscopy (iSCAT) has been developed as a highly sensitive detection technique that visualizes small AuNPs and even unlabeled proteins ( 41 , 42 , 43 , 44 ). Dependence of the iSCAT signal on the radius of AuNP is R 3 because of interference between scattered light from the sample and the reference beam, such as light reflected from the glass surface.…”

Interferometric Scattering Microscopy for the Study of Molecular Motors

“…When illuminated with blue light (<500 nm), an actin filament generates ~1% iSCAT contrast, while a single microtubule produces ~8% contrast, comparable with a 20-nm gold nanoparticle (Andrecka et al, 2016; Ortega Arroyo et al, 2014). Complete immobilization of actin and microtubules on the glass surface is paramount to success when running single-molecule motor assays with high spatiotemporal precision.…”

“…Interferometric detection leads to a lesser drop in scattering contrast with decreasing particle size compared to dark-field microscopy, simplifying the detection of very weak scatterers. In comparison to fluorescence, iSCAT suffers from much poorer background rejection ability, but in exchange can completely remove the need for any fluorescent labeling, including labeling of actin filaments and microtubules, which themselves generate clear iSCAT signals (Andrecka, Arroyo, Lewis, Cross, & Kukura, 2016; Ortega Arroyo et al, 2014). As a result, the relevant single-molecule assays are in many instances easier to implement as they require fewer labeling steps.…”