Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)

Mary Williard Elting,Michael J Levene,James A Spudich,Jonas Korlach,Adam E Cohen,L Stirling Churchman,Sabrina R Leslie,Christopher M J Mcfaul,Jason S Leith

doi:10.1364/oe.21.001189

Abstract

Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.

Highlights

Reducing background fluorescence from fluorophores in solution presents a challenge to many single-molecule fluorescence experiments
We have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments
Though linear zero-mode waveguides (ZMWs) may produce somewhat higher levels of background than have been demonstrated with circular ZMWs, due to their larger excitation volume in the linear dimension, they are still likely to allow single-molecule imaging with ~30-fold higher concentrations than Total internal reflection fluorescence (TIRF)

Summary

Introduction

Reducing background fluorescence from fluorophores in solution presents a challenge to many single-molecule fluorescence experiments. For single-molecule fluorescence imaging of surface-immobilized species, total internal reflection fluorescence (TIRF) microscopy is usually used to interrogate a thin layer of molecules close to the surface in order to suppress background fluorescence from molecules freely diffusing in solution. By coupling to fluorophores using an evanescent field, TIRF restricts the excitation volume to a layer of molecules within a few hundred nanometers above the coverslip surface, enabling individual fluorophores attached to the surface to be resolved in the presence of freely diffusing fluorophores at concentrations up to approximately 100 nM [1,2], many times higher than is possible using epifluorescent excitation. The development and application of single-molecule imaging techniques that probe low affinity interactions inaccessible to TIRF microscopy remains an outstanding challenge to experimental biophysics

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Conclusion