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

Abstract

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.

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

“…Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15,16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community.…”

Open-frame system for single-molecule microscopy

“…This is particularly useful for experiments performed using CLiC microscopy where interferometry (direct imaging of the exciting laser) is used to measure the chamber's height profile. [20][21][22][23] …”

“…[6][7][8][9][10][11][12][13][14][15] In this section, we outline a modular device which extends the capabilities of our microscope to single-molecule microscopy using CLiC. [20][21][22][23] CLiC imaging is based on a simple working principle: confining molecules to a thin sample chamber to allow for single-molecule imaging. CLiC microscopy is performed when a curved optical lens (referred to as the "push-lens") pushes into and deforms the top coverslip of a flow cell.…”

Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

“…These experiments often require additional considerations to prevent the high concentrations of NTPs required (~µM) from generating an excessive fluorescence background signal. To address this challenge, techniques have been developed that utilize zero mode waveguides to confine the detection area to a small volume around a molecule of interest (Eid et al 2009; Elting et al 2013). Arguably the most direct observation of enzyme catalysis is permitted by fluorogenic substrates or cofactors (Fig.…”