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Scanning and Image Reconstruction Techniques in Confocal Laser Scanning Microscopy

Taking advantage of the laser scanning methodology, confocal microscopy has been widely applied in cutting-edge biological research for its three-dimensional, high-resolution imaging capability (Pawley 1995). The principles of confocal were put forward by Marvin Minsky in 1955 (Minsky 1988), yet the experimental demonstration was not accomplished until 20 years later by Cremer brother (Cremer, Cremer et al. 1978), and Brakenhoff et al. (Brakenhoff, Blom et al. 1979). In confocal microscopy, one or more focal spots are scanned relative to the specimen, to extract the signal from the focal spots. The 3-D image can then be reconstructed digitally. Contrary to mechanically scanning the specimen, which is relatively slow as the specimen has to be moved, the laser provides fast scanning and imaging speed for confocal microscopy and therefore is routinely used in commercial state-of-the-art confocal systems. Several laser scanning mechanisms have been employed in confocal microscopy: 1. single spot laser scan with galvanometers; 2. single spot laser scan for real-time imaging and endoscopy; 3. line scan confocal with a line detector or a 2-D detector; 4. multiple spot scanning with 2-D detectors. The single spot galvanometric scan is the most straightforward laser scanning mechanism, which provides an imaging speed of ~1 frame(s) per second (fps) for a typical 512 x 512 image, and is widely used in many confocal laser scanning systems. However, the relatively slow scanning speed of the galvanometer commonly used in a confocal laser scanning microscopy can dramatically limit the system performance in scanning speed and image quality if the data collection is simply synchronized to the galvanometric scanning. In Section 2 we discuss the command and data processing techniques for galvanometric scanning, for example, pixel delay and interlace line switching can be applied on bidirectional scans to cancel aliasing; pixel binning and/or image average can be used to improve the signal-to-noise level; Acquire-On-Fly scanning can be applied when the signal command is a limiting factor for the imaging speed. Due to the limitations placed on maximum galvanometer scanning speed, real-time confocal microscopy relies on the fast scanning mechanism on its fast axis, such as a resonant scanner or rotational polygon mirror. This is discussed in Section 3. The resonant scanner runs at a fixed frequency with variable amplitude, and scans a temporal sinusoidal pattern.