To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA-1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated mu-calpain demonstrated mu-calpain activation specifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry. CA-1 neurons on day 1 showed sustained activation of mu-calpain, and increased immunostaining for inactivated and activated forms of mu- and m-calpains and for PIP2. Activated mu-calpain and PIP2 were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia-hypoglycaemia in vitro provoked intense Ca2+ mobilization with increased PIP2 immunostaining specifically in CA-1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca2+ and PIP2 breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA-1 neuronal death.

We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.

We propose electron beam excitation assisted optical microscope, and demonstrated its resolution higher than 50 nm. In the microscope, a light source in a few nanometers size is excited by focused electron beam in a luminescent film. The microscope makes it possible to observe dynamic behavior of living biological specimens in various surroundings, such as air or liquids. Scan speed of the nanometric light source is faster than that in conventional near-field scanning optical microscopes. The microscope enables to observe optical constants such as absorption, refractive index, polarization, and their dynamic behavior on a nanometric scale. The microscope opens new microscopy applications in nano-technology and nano-science.

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The feasibility of a fiber-optic plate (FOP) microscope system employing a bundle of optical fibers and videomicroscopy for in vivo experiments was investigated. The FOP used here consisted of optical fibers 3 microns in diameter. By inserting the FOP into an animal, optical signals from the deep-lying tissue invisible from the surface could be obtained as two-dimensional images. Using this system, hippocampal cells stained with a fluorescent dye in an anesthetized rat were visualized. Elevation of intracellular free calcium concentration ([Ca2+]i) in the hippocampus of the rat during anoxic exposure was also detected with a fluorescent indicator dye. These results showed that the FOP microscope system was sufficiently applicable to in vivo experiments for studying tissue structure and physiological activity even in the deep regions with fluorometric techniques.