Now, the importance of Ca²⁺ in the cell has been well recognized. However, year of 1983, when we wanted to measure of intracellular Ca²⁺ ion（[Ca²⁺]i）in a single cell such as neuronal cells, we could not find any reliable method. Only one possibility was to use a fluorescence Ca²⁺ indicator（Ca²⁺ chelator）quin-2, which was invented by RY Tsein, who loaded the reagent into cells by modifying the reagent cell permeable form using esterification. They showed successful measurement of [Ca²⁺]i in cell suspension of lymphocytes by a fluorescence spectrophotometer. In 1985 RY Tsein developed a new indicator called fura-2, which had lower affinity to Ca²⁺ than quin-2. The reagent encouraged us to utilize it for measuring [Ca²⁺]i of the neuronal cell. Since neuronal cells might have diversity in their excitability, we should deal with them separately as a single cell. Thus we intended to develop a new method to measure [Ca²⁺]i in single cells by using a fluorescence microscope. Although we encountered so many obstacles before establishing our method, we conquered them and succeeded in developing a primitive, but efficient [Ca²⁺]i measurement system. The method detected the microscopic fluorescence image obtained by a high sensitive video camera. We measured the brightness of the video image using optical fibers and integrating them and made them measurable data. We succeeded in detecting the [Ca²⁺]i. increase in cultured hippocampal neurons during exposure to glutamate in 1986. This was the first report on the real time measurement of Ca²⁺ increase in a single neuronal cell. We improved our method little by little and five years later we introduced an image-processing system. This method became a standard Ca²⁺ imaging system in Japan. We applied this method and found many important Ca²⁺ related biological functions. Recently, we can use a high-performance computer for analyzing the image data and high-capacity memory system for image analysis. As a fluorescence image detector, we can use a multiphoton microscope. Moreover, the molecular biological technique established new Ca²⁺ indicators called GCaMP family by modifying green fluorescence protein. Now these methods are working at the front of biosciences.

Changes in the amount and physical properties of dental plaques were investigated in the oral cavity in which the oral fungal population was maintained at a low level by the long-term use of an antifungal agent as a mouthwash. For the antifungal agent used as a mouthwash, amphotericin B （AMPH-B）, which specifically acts on fungi without influencing bacteria and is unlikely to be absorbed from the oral cavity or gastrointestinal mucosa, was selected. For the index of the fungal level in the oral cavity, Candida indigenous to the oral cavity was selected. We paid attention to time-course changes in the Candida count and the amount of dental plaques and its physical properties observed by palpation using a dental probe and macroscopically.

The long-term use of the AMPH-B-containing mouthwash for 4 months or longer reduced the oral Candida count with time, confirming that the count can be maintained at a low level by continued use of the mouthwash. In addition, dental plaques macroscopically decreased as the Candida count decreased, and the physical properties the dental plaques also changed. Although the quantitative and qualitative influences of fungi in the oral cavity on dental plaques have not been investigated, it was clarified that long-term gargling with AMPH-B reduced the oral fungal level, which may have quantitatively and qualitatively changed dental plaques, i.e., microorganisms constituting the indigenous microbial flora in the oral cavity.

Dental plaques show latent pathogenicity. In addition to the fact that causative microorganisms of the 2 major oral diseases, dental caries and periodontal disease, are present in dental plaques, the causative microorganisms of aspiration pneumonia, which is a major issue in the elderly, are also present. Thus, reduction of the amount of dental plaques may be beneficial for the health of the oral cavity as well as the whole body.

This study was investigated that in order to interaction of cardiovascular effects for adrenaline and hypotension agents, cardiac function was measured for adrenaline administered to blood pressure controlled SHR/Izm by nicardipine or esmolol. Under 5mg/kg/h propofol infusion, arterial blood pressure catheter and P-V catheter were inserted to SHR/Izm. Four hundred micro g/kg/min esmolol or 1 micro g/kg/min nicardipine were infused and 20 micro g/kg adrenaline injected to tongue. Then there were measured blood pressure, pulse rate, stroke volume continuously. Systolic and diastolic pressure elevated from 5 min to 20 min on esmolol. There were significant differences between esmolol and nicardipine at 20 min of systolic, 20min and 30min of diastolic pressure. Pulse rate increased from 5 min to 30 min on nicardipine, there were significant differences between nicardipine and esmolol at 20 min. Stroke volume increased at 10 min, 20 min, 30 min on nicardipine、there were significant differences between esmolol and nicardipine at 5 min, 10 min, 20 min, 30 min. Blood pressure elevated when adrenaline injected to tongue of esmolol administered SHR/Izm, pulse rate and stroke volume increased when adrenaline and nicardipine administered.

Microscopic examination of fixed smear samples are useful in the diagnosis of oral candidiasis as yeast and hyphal morphotypes can be differentiated. While the utility of examination of fixed smears using a fluorescent stain is recognized, this method requires the use of a fluorescence microscope.

Up to now, wide spread use of conventional fluorescent microscopes have been limited as it requires the use of high pressure mercury vapor lamps which are costly with a short life, a dark room and a high voltage supply.

In order to overcome these disadvantages, a LED long-life light source for epi-fluorescence microscopy has been developed. With the mounting of a LED module (Lumin^TM, LW Scientific, Lawrenceville, GA, USA) converting our bright-field microscope for both bright field and epi-fluorescence microscopy, hyphae were clearly observed after fluorescence staining (Fungiflora Y (Biomate, Tokyo, Japan). The methodology used and results are described.