Brillouin scattering microscopy is the only potential tool to realize microscopic mapping of mechanical property in multicellular system (i.e. colony, tissue) with sub-cell resolution. We built a laser-scanning Brillouin microscope system for our biological study on spatial heterogeneity of stiffness in multicellular system. High-numerical-aperture (NA) objective lens (NA ≥ 0.7) and a dual-VIPA based spectrometer were employed to achieve high spatial resolution and high sensitivity, respectively. Addition of a spatial filter at a Fourier plane of the EMCCD detector surface effectively rejected strongly reflected excitation light without loss of Brillouin scattering signal, which accordingly allowed us to observe cells just above glass substrate surface. We performed three-dimensional imaging of Brillouin scattering to see three-dimensional stiffness distribution within a multicellular system. It was found that cells in relatively central region or in close vicinity of glass substrate have higher stiffness, which agrees to biological prediction. We also found presence of anomaly cells with much higher elasticity than surrounding cells. The stiffness imaging was applied to various kinds of multicellular systems including ES cell colonies upon differentiation and artificial tissue grown from iPS cells. The results certified the effectiveness of Brillouin scattering microscope in mechanobiology, developmental biology and regenerative medicine. Several practical issues for biomedical application will also be discussed.
Optical microscopy is an indispensable tool for medical and life sciences. Especially, the microscopes utilized with scattering light offer a detailed internal observation of living specimens in real time because of their non-labeling and non-invasive capability. We here focus on two kinds of scattering lights, Raman scattering light and second harmonic generation light. Raman scattering light includes the information of all the molecular vibration modes of the molecules, and can be used to distinguish types and/or state of cell. Second harmonic generation light is derived from electric polarity of proteins in the specimen, and enables to detect their structural change. In this conference, we would like to introduce our challenges to extract biological information from those scattering lights.
When Raman scattering is excited from the evanescent light field created by illuminating the apex of a sharp metallic
nano-tip, it achieves new aspects with strong enhancement of scattering efficiencies and super resolving capabilities. The
primary mechanism of tip-sample interaction is electromagnetic, which is based on the excitation of localized surface
plasmon polaritons. However, when the tip is close enough to the sample, typically at molecular distances, the chemical
interactions between the tip and the sample become important. Strong temporal fluctuations of Raman scattering,
including fluctuations of peak frequencies and peak intensities, together with extraordinary enhancement of several
peaks, were observed. These temporal fluctuations, which are typical signature of single molecule detection, were
attributed to the changes of molecular orientations of the sample molecules in the upper layer of the nanocluster, which
got chemically adsorbed at the tip molecules.
Near-field Raman spectroscopy using an apertureless metallic probe has attracted much attention owing to its capability of chemical analysis with high spatial resolution far beyond the diffraction limit. The local plasmon excited at the probe tip amplifies optical near-field in the vicinity of the tip apex, and the local field is used to enhance Raman scattering. The metallic probe contributes not only to the enhancement of the Raman scattering, but also to spectral changes due to the chemical and mechanical interaction between the metal and the molecules. We experimentally and theoretically investigated these two effects in this study. These effects selectively provide vibrational information of the molecules directly adsorbed on the metal, and, therefore, have a potential to improve the spatial resolution. In addition, the metallic probe has also been applied for enhancement of nonlinear Raman scattering. Coherent anti-Stokes Raman scattering (CARS) has been strongly enhanced by the probe, and has provided molecular-vibration images of deoxyribo nucleic acid (DNA) with high spatial resolution.
Optical microscopy that can visualize the molecular vibration with a nanometric spatial resolution has been realized by a combination of near-field optics and coherent anti-Stokes Raman scattering (CARS) spectroscopy. A metallic probe with a sharp tip is used to strongly enhance optical near-field in the local vicinity of the tip owing to the excitation of local surface plasmon polariton. CARS signals of molecules in the local area can be strongly induced by the plasmonic field. We have visualized DNA molecules and single-walled carbon nanotubes (SWNTs) with a spatial resolution far beyond the diffraction limit by the tip-enhanced near-field CARS microscopy.
A metallic nano-probe has locally induced coherent anti-Stokes Raman scattering (CARS) of adenine molecules in a nanometric DNA network structure. The excitation fields and CARS polarization are enhanced by the tip apex of the nano-probe through the excitation of local surface plasmons. Owing to the third-order nonlinearity, the excitation of the CARS polarization is extremely confined to the end of the tip apex, resulting in the spatial resolution far beyond the diffraction limit of light. Our CARS microscope using a silver-coated probe visualized the DNA network structure at a specific vibrational frequency (~1337 cm<sup>-1</sup>) of adenine molecules with a spatial resolution of ~15 nm and sufficient sensitivity.
A light microscope capable to show images of molecules in nanometer scale has been a dream of scientists, which, however, is difficult due to the strict limitation of spatial resolution due to the wave nature of light. While there have been attempts to overcome the diffraction limit by using nonlinear response of materials, near-field optical microscopy could provide better detecting accuracy. In this paper, we present molecular distribution nano-imaging colored by Raman-scattering spectral shifting, which is probed with a metallic tip. The metallic probe tip has been used to enhance the optical field only in the vicinity of probe tip. The effect is similar to the one seen in the detection of molecules on the metal-island film, known as surface-enhanced Raman spectroscopy (SERS), while in this case a single metallic tip works for the field enhancement in nanometer scale.