Optical microscopy enables us to observe motions of protein molecular motors as they work in the biological systems. By labeling molecules of interest with appropriate probes, the motions of individual molecules can be tracked. Localization precision (how precisely we can determine the position) of moving molecule is primary determined by the photon numbers captured by a single image frame. One nanometer localization precision and millisecond time resolution can be achieved with fluorescent probes, by capturing ~10,000 photons. Although further improvement of the localization precision is crucial for deeper understanding of the operation mechanisms of molecular motors, limited number of photons obtained from the fluorescent probe have restricted the improvement. Recently, gold nanoparticles (AuNPs) which strongly scatter the incident light, have been used as an alternative of the fluorescent probes (Fig. 1). In previous studies, microsecond time resolution has been achieved with AuNPs. However, the fundamental limit of the localization precision has not been experimentally investigated in detail.
In this study, by using newly developed annular illumination total internal reflection dark-field microscopy, Ando and co-workers in Institute for Molecular Science, Japan, succeeded in achieving atomic-level, 1.3 angstrom (Å) localization precision with 40 nm AuNPs at 1 millisecond time resolution (Fig. 2). Furthermore, even at 33 microsecond time resolution, 5.4 Å localization precision has been successfully achieved.
The authors firstly investigated the fundamental law which limits the localization precision of AuNP, and confirmed that the localization precision is improved in proportion to the square root of the photon number, as previous theoretical considerations. Furthermore, the lower limit of the localization precision with a dark-field imaging system previously developed by the authors was around 3 Å, which was restricted by signal saturation of the detector. To improve the localization precision further, authors newly developed an annular illumination total internal reflection dark-field microscopy. In this system, by shaping the laser light into a ring, higher laser intensity than the previous system can be used without damaging the objective lens. Furthermore, smaller pixel size of the detector (larger number of pixels in an image) was applied to suppress the signal saturation. These improvements enable to achieve 1.3 Å localization precision at 1 millisecond time resolution.
Furthermore, the authors investigated relationship between localization precision and time resolution in detail. The developed system achieved 5.4 Å localization precision even at 33 microsecond time resolution. To minimize possible steric hindrances of AuNPs on the protein molecular motors, the authors also investigated size dependence on the localization precision, and achieved 1.9 Å localization precision at 1 millisecond time resolution with 30 nm AuNPs.
Then, by using the developed imaging system, the authors observed stepping motions of a dimeric linear molecular motor kinesin, moving along the microtubule in detail (Fig. 3). One motor domain (head) of the kinesin was labeled by 40 nm AuNP, and the motion was captured at 10 microsecond time resolution. In a previous study, unbound head showed diffusional motion only at right side of bound head on microtubule. This result implies unidirectional rotation of two heads of kinesin during linear motion. High-localization, high-speed single-particle tracking performed in this study successfully revealed details of the transition from bound to unbound states of the kinesin head. Because no apparent leftward trails were observed, the authors concluded that the kinesin actually rotates unidirectionally during the linear motion.
Not only kinesin, the technique developed in this study will capture dynamics of various protein molecular motors with atomic-level localization precision and microsecond time resolution. Indeed, the authors recently resolved the forward and backward 1-nm steps during fast unidirectional motion of a chitinase driven by processive catalysis, and revealed that processive chitinase operates as a "burnt-bridge" Brownian motor. Thus, the developed system will largely contribute to further understandings of the operation mechanisms of many protein molecular motors. Furthermore, the developed system can be also applied to visualize atomic-level motions of synthetic molecular motors, which are much smaller than the protein molecular motors.