Gold nanorods (AuNRs) is a good imaging probes for translational and rotational tracking because of various properties, such as non-photobleaching, non-blinking as well as its strong light-scattering at its localized surface plasmon resonance (LSPR). Rotational information can be obtained by looking at intensity or patterns of the AuNRs. One of the current methods of imaging plasmonic nanoparticles are differential interference contrast microscopy (DIC), which looks at the intensity of AuNR. However, this method suffers from angular degeneracy since it can only resolve 0-90° azimuth angle instead of full range. Pattern recognition method, i.e., defocused dark field microscopy, suffers from high defocus distance sensitivity. Slight changes in z-position can cause the defocus pattern to deform. Here, we developed a multi-channel, multi-modality imaging system that enables us to simultaneously obtain multicolor fluorescent images and (de)focused dark field images of AuNRs. The (de)focused dark field images provide us with full-range azimuth angle, polar angle as well as 3D displacement information of the AuNRs. Parallax was included in this method and it kept AuNRs in-focus in the focused channel, while it also maintains the same defocused distance for the AuNRs in the defocused channel during single particle orientational and rotational tracking (SPORT) experiments. We used this method to study dynamin fission of clathrin-coated vesicle in the final stage of clathrin-mediated endocytosis (CME). Not only the rotation time, step-azimuth and polar angle were identified, a large quick twist named as the super twist before fission of the clathrin-coated vesicle were discovered in all observed cases. Using this method, new light can be shed on different kinds of dynamic processes involving various functional proteins inside cells.

Multi-channel, multi-modality single particle orientational and rotational tracking

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ACS Fall 2022

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