Design Of The Fluorescent Microscopy System To Detect [Ca～(2+)]_i In Living Cells

As the important intracellular secondary messenger, Ca2+ regulates many physiological activities of cells. Fluorescence microscopy technique is suitable to detect [Ca2+]i in living cells because it has the short responding time and is innocuous to living cells. Therefore, the design for the fluorescent microscopy system to detect [Ca2+]i in living cells is very helpful to the research of physiology and biology. This system provides a new window to observe the intracellular activity.Our primary work is to build the fluorescent microscopy system to detect [Ca2+]i in living cells. In this system, the light from the xenon light source is converged by elliptical mirror and diffracted by diffraction grating; then the lightguide and dual-coupling condensor transmit monochromatic light to the specimen plane to generate the fluorescence of the fluorescent probe within the cell; at last the fluorescent signal is detected by the photomultiplier tube.The main content of this paper could be divided to four sections as follows:The first part concerns in the principle and structure of monochromator. The key component of its optical sketch is the elliptical mirror, not the torodial mirror, which is normally used in the foreign familiar products and more expensive than the elliptical mirror. In the second part, a novel dual-coupling condensor is presented. To maintain optimal throughput and guarantee optimal homogeneity in the specimen plane, we designed suitable structure of condenser, carefully corrected aberrations and used dichroic beamsplitters to keep the proper proportion of the incident light energy.The following part mentions the equipment to detect the faint fluorescent signal. The adjustable amplifier and filter are included in the equipment.In the last part, the installation process and debug strategy of this system is introduced. Using the system, the dynamic curve of [Ca2+]i in PC12 cells after high K+ depolarization is given.