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Proceedings Paper

Label-free observation of tissues by high-speed stimulated Raman spectral microscopy and independent component analysis
Author(s): Yasuyuki Ozeki; Yoichi Otsuka; Shuya Sato; Hiroyuki Hashimoto; Wataru Umemura; Kazuhiko Sumimura; Norihiko Nishizawa; Kiichi Fukui; Kazuyoshi Itoh
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Paper Abstract

We have developed a video-rate stimulated Raman scattering (SRS) microscope with frame-by-frame wavenumber tunability. The system uses a 76-MHz picosecond Ti:sapphire laser and a subharmonically synchronized, 38-MHz Yb fiber laser. The Yb fiber laser pulses are spectrally sliced by a fast wavelength-tunable filter, which consists of a galvanometer scanner, a 4-f optical system and a reflective grating. The spectral resolution of the filter is ~ 3 cm-1. The wavenumber was scanned from 2800 to 3100 cm-1 with an arbitrary waveform synchronized to the frame trigger. For imaging, we introduced a 8-kHz resonant scanner and a galvanometer scanner. We were able to acquire SRS images of 500 x 480 pixels at a frame rate of 30.8 frames/s. Then these images were processed by principal component analysis followed by a modified algorithm of independent component analysis. This algorithm allows blind separation of constituents with overlapping Raman bands from SRS spectral images. The independent component (IC) spectra give spectroscopic information, and IC images can be used to produce pseudo-color images. We demonstrate various label-free imaging modalities such as 2D spectral imaging of the rat liver, two-color 3D imaging of a vessel in the rat liver, and spectral imaging of several sections of intestinal villi in the mouse. Various structures in the tissues such as lipid droplets, cytoplasm, fibrous texture, nucleus, and water-rich region were successfully visualized.

Paper Details

Date Published: 22 February 2013
PDF: 9 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 858806 (22 February 2013); doi: 10.1117/12.2002772
Show Author Affiliations
Yasuyuki Ozeki, Osaka Univ. (Japan)
PRESTO, Japan Science and Technology Agency (Japan)
Yoichi Otsuka, Canon Inc. (Japan)
Shuya Sato, Canon Inc. (Japan)
Hiroyuki Hashimoto, Canon Inc. (Japan)
Wataru Umemura, Osaka Univ. (Japan)
Kazuhiko Sumimura, Osaka Univ. (Japan)
Norihiko Nishizawa, Nagoya Univ. (Japan)
Kiichi Fukui, Osaka Univ. (Japan)
Kazuyoshi Itoh, Osaka Univ. (Japan)

Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)

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