COMBINED ANALYSIS OF INTRACELLULAR CALCIUM WITH DUAL EXCITATION FLUORESCENCE PHOTOMETRY AND IMAGING

We have developed an integrated microscopy system combining fast dual- excitation fluorescence photometry and digital image analysis with hig h spatial resolution, based mainly on standard components. With the co mbination of these well-established techniques in one setup it is poss ible to monitor intracellular calcium with both sufficiently high temp oral and high spatial resolution on the same preparation for many biol ogical applications, Our system consists of a commercially available d ual-excitation photometric system, an attached intensified charge coup led device (ICCD) camera, and a frame grabber board. With this integra ted setup one can easily switch between the fast photometric mode (v(r atio) = 100 Hz) and the imaging mode (v(ratio) = 4.17 up to 17 Hz). We used the system to record Fura-2 calcium images (340/380 nm ratios), which were correlated with the faster spot measurements and were analy zed by means of image processing, As an example for its application we reconstructed caffeine-induced calcium transients released from the s arcoplasmic reticulum of isolated and permeabilized skeletal muscle fi ber preparations. Such a combined technique will also be important for cellular studies using other fluorescence indicators, Additionally, t he described system has an external trigger facility that enables comb ination with other cell physiological methods, e.g., electrophysiologi cal techniques.