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SPIE BiOS, 2013, San Francisco, California, United States, Yasuyuki Ozeki, Yoichi Otsuka, Shuya Sato, Hiroyuki Hashimoto, Wataru Umemura, Kazuhiko Sumimura, Norihiko Nishizawa, Kiichi Fukui, Kazuyoshi Itoh, "Label-free observation of tissues by high-speed stimulated Raman spectral microscopy and independent component analysis," Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 858806 (22 February 2013); https://doi.org/10.1117/12.2002772, 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.

Ozeki Y., Kitagawa Y., Sumimura K., et al. Stimulated raman scattering microscope with shot noise limited sensitivity using subharmonically synchronized laser pulses. Optics Express, 18, 13, 13708. https://doi.org/10.1364/OE.18.013708., We propose and demonstrate the use of subharmonically synchronized laser pulses for low-noise lock-in detection in stimulated Raman scattering (SRS) microscopy. In the experiment, Yb-fiber laser pulses at a repetition rate of 38 MHz are successfully synchronized to Ti:sapphire laser pulses at a repetition rate of 76 MHz with a jitter of <8 fs by a two-photon detector and an intra-cavity electro-optic modulator. By using these pulses, high-frequency lock-in detection of SRS signal is accomplished without high-speed optical modulation. The noise level of the lock-in signal is found to be higher than the shot noise limit only by 1.6 dB. We also demonstrate high-contrast, 3D imaging of unlabeled living cells.

Hieu M. Dang, Gen Omura, Toshiyuki Omano, Masatomo Yamagiwa, Shin'ichiro Kajiyama, Yasuyuki Ozeki, Kazuyoshi Itoh, and Kiichi Fukui "Label-free imaging by stimulated parametric emission microscopy reveals a difference in hemoglobin distribution between live and fixed erythrocytes," Journal of Biomedical Optics 14(4), 040506 (1 July 2009). https://doi.org/10.1117/1.3207151, We demonstrate that stimulated parametric emission (SPE) microscopy enables label-free, 3-D visualization of internal hemoglobin distribution of live mouse and chicken erythrocytes with high sensitivity. Change in hemoglobin distribution in chicken erythrocytes before and after ethanol fixation is clearly visualized. © 2009 Society of Photo-Optical Instrumentation Engineers.

Ozeki Y., Dake F., Kajiyama S., et al. Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy. Optics Express, 17, 5, 3651. https://doi.org/10.1364/OE.17.003651., We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.

Isobe K., Ozeki Y., Kawasumi T., et al. Highly sensitive spectral interferometric fourwave mixing microscopy near the shot noise limit and its combination with two-photon excited fluorescence microscopy. Optics Express, 14, 23, 11204. https://doi.org/10.1364/OE.14.011204., We present spectral interferometric four-wave mixing (FWM) microscopy with a nearly shot-noise limited sensitivity and with the capability of separating FWM signals from fluorescence signals. We analyze the requirements for obtaining the shot-noise limited sensitivity and experimentally achieve the sensitivity that is only 4-dB lower than the shotnoise limit. Moreover, we show that only FWM signals can be extracted through the Fourier filtering even when the FWM spectrum is overlapped and overwhelmed by the fluorescence spectrum. We demonstrate simultaneous acquisition of FWM and two-photon excited fluorescence images of fluorescent monodispersed polystyrene microspheres.