SAKAI Masahiro Department of Anatomy, Kyoto University, Instructor, 医学部, 助手 (40183363)
FUJIMOTO Kazushi Department of Anatomy, Kyoto University, Lecturer, 医学部, 講師 (50159125)
FUJIMOTO Toyoshi Department of Anatomy, Kyoto University, Associate Professor, 医学部, 助教授 (50115929)
|Budget Amount *help
¥23,500,000 (Direct Cost : ¥23,500,000)
Fiscal Year 1991 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1990 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 1989 : ¥15,000,000 (Direct Cost : ¥15,000,000)
In order to establish a new method for an electron microscopic cytochemical analysis, we developed an analytical color fluorescence electron microscope(ACFEM)and applied this method to the biomedical research. On the basis of a high resolution scanning electron microscope, the ACFEM, equipped with a cathodoluminescence(CL)-collecting mirror and a spectral multichannel analytical system, enables us to obtain color CL images, monochromatic CL images at any given wavelength, and conventional SEN images, as well as CL spectrum when an electron beam is scanning over the surface of specimens.
Using the ACFEM, we observed CL from a varieties of organs and tissues without fluorescent staining to analyze the substances which emit CL. CL was found to be emitted from lipid droplets(LD)in biological specimens with a two-peak pattern spectrum at 320 nu and 430-450 na respectively. It was proved that the 320 nm-CL was derived from cholesterol esters, because 320 nu-CL, which was usually detected in t
he LD of adrenal cortex, corpus luteum, theca interna and granulosa cells of the rats, disappeared when the animals were treated with hypocholesterolemic agents, and showed an increase of intensity after hypophysectomy. Similarly, the 430-450 na-CL was demonstrated to be derived from a mixture of lipids at least including vitamin A ester by the research dealt with retina and testes of the rats. So far, it was impossible to analyze the components in a single lipid droplet by means of cytochemistry for the reason of lacking antibodies with high specialities against lipids, such as steroids and retinoids. This study methodologically dissolved this problem.
Moreover, we also observed that FITC-IgG emits CL under the ACF24. This fact indicates the possibility to directly introduce fluorescent antibody method to electron microscopy. Recently, the study on evaluating diamonds and developing new photo sensitive materials have revealed that the ACFEM could be very useful in the industrial research fields, also. Less