Color cathodoluminescence images and cathodoluminescence spectra analysis of biological materials.

Abstract

In an attempt to detect cathodoluminescence (CL), an Akashi scanning electron microscope (SEM) ISI-DS130 was remodeled into a cathodoluminescence electron microscope (CEM or fluorescence electron microscope FEM). A newly shaped mirror, combining three half ellipsoidal mirrors, was located over the specimen holder of the top stage for color CL use. The bottom stage was modified with a half ellipsoidal mirror, photomultiplier tube (PMT) and monochrometer, so that not only CL images, but also CL spectra could be obtained.An effective specimen-preparation method, known as the simple cryo-SEM method (24), was devised for CL observation.With these instruments and methods, color CL images and CL spectra of the biological substances were analyzed.Ito cells of vitamin A (vit A)-loaded rat liver and zona fasciculata cells of rat adrenal cortex emitted intense CL from their lipid droplets (LD). In order to identify the origin of this CL emission, analysis of the color CL images and the CL spectra were available.Cathodoluminescence (CL) is a light emission, in the visible, near ultraviolet and infrared regions under the bombardment of an electron beam (31). CL phenomenon is similar to fluorescence or phosphorescence excited by ultraviolet light, but it has some intrinsic advantages over the fluorescence as described by Brocker et al. (6,31) : 1) CL can be amplified and measured quantitatively, 2) The depth of focus and the resolution in the CL mode are higher than in the fluorescence microscope. 3) Some substances exhibit luminescence only under electron irradiation. Furthermore, CL of organic chemicals stems from loosely bound it electrons; it shows the information of the chemical bond. Just as secondary electrons (SE), backscattered electrons, Auger electrons and X-rays are often used for observing or analysing substances under scanning electron microscope (SEM), so CL should become an effective analytical tool.Although CL studies as an electron probe can be traced to 1952, the CL applications to biological and medical fields have not made remarkable advances.

Application of analytical color fluorescence electron microscopy to biomedical field: I. Vitamin A ester in rat retina.

“…To confirm the hypovitaminosis A in vitamin A-deficient dieted rats in experiment 1, unfixed rat livers of this group were examined by the ACFEM according to the method of Itoh et al (19), and the animals which showed no CL emission from liver were used for the experiments. 1.…”

“…In previous studies (19,22), we have developed an analytical color fluorescence electron microscope (ACFEM) based on a high resolution scanning electron microscope (SEM). The ACFEM enabled us to detect CL from biological specimens as a color image simultaneously with secondary electron (SE) images and also to analyze their CL spectra.…”

Application of analytical color fluorescence electron microscopy to biomedical field: II. Cholesterol ester in rat adrenal cortex.

“…This new microscopy is obviously a useful tool in the field of histo-and cytochemistry. Itoh et al (7) previously reported that blue-green CL was observed in the rat adrenal cortex, however they did not succeed in identifying the origin of this CL, because CL emission of adrenal cortex changes quickly. Koike et al (9) reported that several intermediary metabolites for the steroid hormone synthesis could be separated by CL spectroscopy in vitro by using a photon counting method.…”

“…In previous studies, we have developed an analytical color fluorescence electron microscope (ACFEM) (7,9,15), which enabled us to observe color CL images of biological materials simultaneously with corresponding secondary electron (SE) images and also to analyze CL spectra. Using the ACFEM, we could reveal the distribution of vitamin A ester in the rat eye by a combination of a cryo-SEM method and spectroscopic CL analysis as shown in the accompanying paper (15).…”

Effect of hypophysectomy on rat adrenocortical cells studied by analytical color fluorescence electron microscopy.

“…However, by means of an analytical color fluorescence electron microscope (ACFEM) developed recently, it is possible to localize cholesteryl ester in lipid droplets (LD) directly. Using the ACFEM, cathodoluminescence (CL) from the adrenocortical cells have been investigated (2,3,5).…”