A low-light level video microscope with long working distance objective lenses has been built as part of our integrated three-dimensional (3-D) light microscopy workstation (Fig. 1). It allows the observation of living specimens under sufficiently low light illumination that no significant photobleaching or alternation of specimen physiology is produced. The improved image quality, depth discrimination and 3-D reconstruction provides a versatile intermediate resolution system that replaces the commonly used dissection microscope for initial image recording and positioning of microelectrodes for neurobiology. A 3-D image is displayed on-line to guide the execution of complex experiments. An image composed of 40 optical sections requires 7 minutes to process and display a stereo pair.The low-light level video microscope utilizes long working distance objective lenses from Mitutoyo (10X, 0.28NA, 37 mm working distance; 20X, 0.42NA, 20 mm working distance; 50X, 0.42NA, 20 mm working distance). They provide enough working distance to allow the placement of microelectrodes in the specimen.
