Construction of a confocal microscope for real-time x-y and x-z imaging

We describe the construction of a simple 'real-time' laser-scanning confoca l microscope, and illustrate its use for rapid imaging of elementary intrac ellular calcium signaling events. A resonant scanning galvanometer (8 kHz) allows x-y frame acquisition rates of 15 or 30 Hz, and the use of mirrors t o scan the laser beam permits use of true, pinhole confocal detection to pr ovide diffraction-limited spatial resolution. Furthermore, use of a piezoel ectric device to rapidly focus the objective lens allows axial (x-z) images to be obtained from thick specimens at similar frame rates. A computer wit h image acquisition and graphics cards converts the output from the microsc ope to a standard video signal, which can then be recorded on videotape and analyzed by regular image processing systems. The system is largely made f rom commercially available components and requires little custom constructi on of mechanical parts or electronic circuitry. It costs only a small fract ion of that of comparable commercial instruments, yet offers greater versat ility and similar or better performance. (C) Harcourt Publishers Ltd 1999.