title: Raman and SERS microscopy for molecular imaging of live cells.
abstract: Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2'-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in ∼8 h.
authors.affiliation: Department of Applied Physics, Osaka University, Suita, Osaka, Japan.
authors.forename: Almar F
authors.initials: AF
authors.lastname: Palonpon
authors.forename: Jun
authors.initials: J
authors.lastname: Ando
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamakoshi
authors.forename: Kosuke
authors.initials: K
authors.lastname: Dodo
authors.forename: Mikiko
authors.initials: M
authors.lastname: Sodeoka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kawata
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Fujita
mesh_terms: D001692:Biological Transport; D018929:Cell Culture Techniques; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D055095:Optics and Photonics; D012984:Software; D013059:Spectrum Analysis, Raman / Q000295:instrumentation / Q000379:methods*
chemical_list: D006046:Gold