Cathodoluminescence (CL) is the light that is emitted from a material irradiated by an electron beam. The present study was undertaken to show the applicability to biological studies of a scanning electron microscope (SEM) equipped with a high-sensitive cathodoluminescence detection system. For this purpose, we injected inorganic fluorescent powders (P43) suspended in phosphate buffered saline into rat blood circulation, fixed the animals with glutaraldehyde within a day, and observed the hepatic tissues with a SEM. Our instrument enabled the simultaneous collection of both secondary electron (SE) and CL images of these tissues. Backscattered electron (BSE) images of the same portion were also able to be obtained with this microscope. SE and BSE images clearly showed the three-dimensional structure of the hepatic tissues including hepatocytes, Kupffer cells, Ito cells, and sinusoidal epithelial cells, while CL images visualized cathodoluminescence signals emitted from P43 as bright spots. We observed non-coated tissues under a low-vacuum condition and metal-coated tissues under a high-vacuum condition, and found that the high-vacuum observation of metal-coated tissues provided high quality CL images of P43 in the Kupffer cells. The superimposition of the CL images onto the corresponding SE or BSE images revealed that bright spots in the CL images were produced by the fluorescent powders uptaken by Kupffer cells. These findings indicate that the detection of CL as well as SE or BSE signals by SEM all provide us with useful information on the distribution of fluorescent tracers in tissues and cells in three-dimensional images.

In the present study, we successfully obtained nonstaining blood flow images of a developing fish egg embryo using optical interference caused by the Doppler shift. The spectral distribution of light reflected by moving objects such as the heart and red cells was found to be different from that of the incident light because of the Doppler effect. Interference between different frequency components was observed in an interferogram through heterodyne interaction using an imaging-type two-dimensional Fourier spectroscopic system, and information on the intensities of the spectral components was obtained by Fourier transformation. Beat signals with specific frequencies due to the heart beating and blood flow of the fish egg embryo were detected. When the signals were plotted in two dimensions, the heart part and vessel flows were clearly visualized without staining. In addition, near-infrared (NIR) images were produced using absorbance spectra of the molecular vibrations of O-H and C-H groups included in water, hydrocarbons, and aliphatic compounds. Obtaining nonstaining blood flow images using heterodyne optical interference and images of molecular distribution using molecular vibrational information simultaneously manifests an exciting advance in NIR imaging.

By combining a bolometer detector with an imaging-type interferometer, an inexpensive, easy-to-handle wide-field mid-infrared hyperspectral imaging apparatus was produced. We measured the distributions of four types of thin adhesive layers on an aluminium plate and analysed the results using correlation coefficients to visualise the distribution of various adhesives that cannot be discerned by the naked eye or conventional methods such as visible/near-infrared spectroscopic/fluorescent photography. The measurement wavelength range, obtained spectrum’s wavenumber resolution, and measurement time was 8–14 μm, about 9 cm−1, and about 30 s, respectively. Using conventional methods, adhesives could not be distinguished from the others. By using this method, we found that adhesives could be precisely distinguished by setting an appropriate threshold value for the correlation coefficient. Thus, our approach can accurately measure the spatial distribution of different types of adhesive that cannot be discriminated by conventional methods.

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