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.