Multicolor High-Speed Tracking of Single Biomolecules with Silver, Gold, and Silver–Gold Alloy Nanoparticles

Gold nanoparticles have been used as imaging probes to track the motions of single biomolecules. To investigate behaviors of various biomolecules simultaneously, increase of the color palette is necessary. Here we developed a multicolor high-speed single-particle tracking system using silver, gold, and silver–gold alloy (5:5 composition ratio) nanoparticles. The peak wavelengths of the plasmon resonances for 30 nm silver, 30 nm silver–gold alloy, and 40 nm gold nanoparticles were around 410, 460, and 530 nm, respectively, and we constructed multicolor total internal reflection dark-field microscope with multiple lasers at 404 nm for silver, 473 nm for silver–gold alloy, and 561 nm for gold nanoparticles. By the use of a spectrophotometer in the detection optics, scattering images at each wavelength were projected onto different portions of a single two-dimensional detector. High-contrast images of silver, silver–gold alloy, and gold nanoparticles were simultaneously obtained in different color channels. After correction of positional shifts among different color channels by affine transformation, a maximum shift less than 17 nm was achieved. Furthermore, an additional 649 nm laser enabled the detection of plasmon coupling by transient dimer formation of two nanoparticles. With this system, diffusional motions of phospholipids in a supported membrane and stepping motions of kinesins along microtubules were successfully observed with a localization precision of 2 nm and a time resolution of 100 μs at each channel. Our method will pave the way to investigate the operation mechanisms of complex biomolecular systems.