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Gold nanoparticles (AuNPs) have been widely used as optical probes to observe motions of biomolecules such as lipids and proteins in biological systems. They efficiently scatter light without photobleaching, and provide high contrast in optical images. Motions of single biomolecules, labeled with AuNPs, have been investigated by tracking center positions of AuNPs in optical image. Nanoscale stepping motions of motor proteins and fast diffusional motions of lipids in membranes have been observed. To understand the working mechanism of tiny and complex biological molecules in detail, further improvement of the localization precision, and imaging capability for multiple biomolecules will be important. We developed an annular illumination total internal reflection dark-field microscope with axicon lens to achieve localization precision at angstrom level and temporal resolution at microsecond order. Localization precisions at 0.3 nm was achieved with 40 nm AuNPs at temporal resolution of 100 µs. We used this system for the observation of bio-molecular motors, such as kinesins and dyneins. We also developed a multicolor single particle tracking system using silver and silver-gold alloy nanoparticles (AgNPs and AgAuNPs) together with AuNPs. We constructed a total internal reflection multicolor dark-field microscope with multiple lasers that match the plasmon resonance wavelength of AgNPs, AgAuNPs, and AuNPs, respectively. A spectrophotometer was used in imaging optics, to project scattering images at each wavelength on the different portions of two-dimensional high-speed CMOS camera. Motions of multiple phospholipids in supported lipid membranes, and multiple kinesins were simultaneously observed at 100 µs time resolution.
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