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.
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Acknowledgements
We thank M. Okada and N.I. Smith for technical support and helpful discussions. This work was partly supported by JST-ERATO, JST-CREST and JSPS KAKENHI grant nos. 24710267 and 23710276.
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A.F.P., J.A. and K.F. developed and constructed the Raman microscope system. A.F.P., H.Y. and J.A. conducted the experiments presented here. K.D., M.S., S.K. and K.F. provided conceptual input and supervised the research. A.F.P., J.A. and K.F. wrote the manuscript with contributions from all other coauthors.
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Supplementary Video 1
Time-resolved observation of SERS from gold nanoparticles in cells. Left: SERS images. Color channels: red, 501 cm−1; blue, 807 cm−1; green, 1143 cm−1. Right: Darkfield images. (MOV 544 kb)
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Palonpon, A., Ando, J., Yamakoshi, H. et al. Raman and SERS microscopy for molecular imaging of live cells. Nat Protoc 8, 677–692 (2013). https://doi.org/10.1038/nprot.2013.030
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DOI: https://doi.org/10.1038/nprot.2013.030
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