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

Abstract

Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ∼0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 normalÅ localization precision for 40 nm AuNPs and 1.9 normalÅ localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 μs time resolution, localization precisions at 5.4 normalÅ for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors.

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

“…chased from British Biocell International Solutions (EMGC30, BBI). The actual diameter was 30.8 ± 2.4 nm, as previously observed with an electron microscope 31 . The procedure for surface modification of 30 nm AuNP with streptavidin was same as previously reported method with small modifications 31 .…”

“…The actual diameter was 30.8 ± 2.4 nm, as previously observed with an electron microscope 31 . The procedure for surface modification of 30 nm AuNP with streptavidin was same as previously reported method with small modifications 31 . In short, the incubation time with alkanethiol mixture solution was changed to 5 h. The time for centrifugation to remove unreacted alkanethiols and streptavidin was changed to 5 min at 10,000 × g.…”

“…Then, we conducted single-molecule imaging of stepping motion at 100 μs time resolution at 1 mM ATP, a physiologically-relevant [ATP]. For imaging of AuNP-labeled dynein, we used annular illumination total internal reflection dark-field microscopy 31 . In our experimental condition, 30 nm AuNP fixed on the glass surface showed localization precision of 0.7 nm at 100 μs time resolution.…”

“…A continuous-wave 532 nm laser (Excel, Laser Quantum, UK) was used for an illumination light source of dark-field imaging. We constructed annular illumination total internal reflection dark-field microscopy with perforated mirror and axicon lens, as reported previously 31 . The ring-shaped beam was formed by introducing laser beam (2020) 10:1080 | https://doi.org/10.1038/s41598-020-58070-y www.nature.com/scientificreports www.nature.com/scientificreports/ into axicon lens (α = 10 °).…”

Damped White Noise Diffusion with Memory for Diffusing Microprobes in Ageing Fibrin Gels

“…Thus, it is important to have a quantitative description of the mechanics and complex structural formation of fibrin as a viscoelastic biomaterial (3, 9–11). Since not much is known about the diffusive behavior of probe particles in fibrin as its structure evolves in time, we use passive microrheology via video microscopy to reveal the gelation dynamics of fibrin by tracking the movement of probe particles suspended in its network of protein polymers (12–14). Moreover, to elucidate how the structure of fibrin is affected at different ageing times, we present a theoretical framework for the stochastic fluctuations exhibited by the experimental observations.…”