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.