The principle of CLSM aims to overcome some limitations of traditional wide-field fluorescence microscopes. In a conventional fluorescence microscope, the entire specimen is flooded evenly in light from a light source. All parts of the sample can be excited at the same time and the resulting fluorescence is detected by the microscope’s photodetector or camera including a large unfocused background part. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane can be detected, the image’s optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes.

CLSM works on the principle of point excitation in the specimen and point detection of the resulting fluorescent signal. The specimen was scanned one point at a time that permits thin optical sections to be collected by simply changing along the z axis (z-focus). The resulting images can be stacked to produce a 3D image of the specimen.