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 confocal microscope, and illustrate its use for rapid imaging of elementary intracellular 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 to scan the laser beam permits use of true, pin-hole confocal detection to provide diffraction-limited spatial resolution. Furthermore, use of a piezoelectric device to rapidly focus the objective lens allows axial (x-z) images to be obtained from thick specimens at similar frame rates. A computer with image acquisition and graphics cards converts the output from the microscope to a standard video signal, which can then be recorded on videotape and analyzed by regular image processing systems. The system is largely made from commercially available components and requires little custom construction of mechanical parts or electronic circuitry. It costs only a small fraction of that of comparable commercial instruments, yet offers greater versatility and similar or better performance.