Characteristics of Confocal Laser Scanning Microscope
If the pinhole is removed from a laser scanning confocal microscope imaging system, the defocused light (light from off focal planes) can reach the detector, the resolution in the depth direction will be greatly reduced, then the non-confocal optical microscope would be no much different from an ordinary microscope. So the most fundamental difference between confocal laser scanning microscope and the ordinary microscope is that there is a pair of conjugate pinholes to limit received light rays only from focal plane, thus largely eliminating the blur effect of defocused light. Another remarkable difference lies on the fact that laser scanning system can acquire an extremely low aberration as the whole optical imaging system needs only to deal with point-to-point imaging, instead of a complete field of view, which requires a sophisticated optical aberration reduction for system design. Compared with a general-purpose optical microscope, a confocal laser scanning microscope has the following characteristics:
- (1) Scattering background of confocal laser scanning microscope is lower than normal optical microscope, resulting in a high image contrast and detection sensitivity.
- (2) Three-dimensional imaging can be reconstructed from layer to layer image data, forming a light-ray tomography through line by line, point by point threedimensional scanning, completely different from computed tomography method. Image data stored in the computer allows display any two-dimensional crosssectional or perspective view of three-dimensional tomography.
(3) Utilization of laser light source provides a small size and high brightness illumination light spot on focal place. Monochromatic laser light essentially eliminates chromatic aberration of the system. With advances in laser technology, more and more types of lasers have been fully able to meet the needs of biomedical research sample.
Commercialization of laser scanning confocal microscope are usually equipped with powerful image processing software, realizing functions of positioning of multiple fluorescent labeling, optical sectioning, 3D reconstruction, time series scanning and other quantitative analysis.