Video-Rate Confocal Microscopy

Most current confocal laser-scanning microscopes require <0.1 sec, more typically ~1 sec, to complete a single full scan of the field of view. Such speeds are considerably slower than conventional video displays or the psychophysical flicker-fusion frequency, so they give the visual impression of discontinuous image acquisition, rather than a smoothly evolving scene. This speed limitation is imposed by the scanning rates of conventional scanners based on linear servo galvanometers, not from any inherent feature of the confocal principle. Scanning at video rate has many advantages. The most obvious but not necessarily the most important is the psychophysical appearance of smooth motion or evolution, which greatly aids the user to locate and focus on regions of interest. Many biochemical signals inside living cells, particularly membrane potential and cytosolic free Ca2+and Na+, undergo changes in the time scale of seconds down to tenths of milliseconds. Such signals are especially important in neurobiology because neurons rely on such biochemistry to perform their special function of fast information processing.