Fundamentals of Confocal Laser Scanning Microscopy
Figure2.1 shows a description of the basic principles of confocal laser scanning microscope imaging. The excitation light goes through illumination pinhole and form a point light source, reflected by a beam splitter by means of exciting filter, focused by the microscope objective into the three-dimensional sample. By scanning in the direction perpendicular to the optical axis in the xy plane (focal plane), fluorescence emitting from illuminated region on the focal plane and on the top and bottom of the focal plane is collected by objective with the beam splitter and emission filter. There is a confocal pinhole in front of the detector, the illumination pinhole and confocal pinhole is conjugate with respect to the focal plane of the objective lens, so that only the fluorescence emitted from the focal plane can be focused to a confocal pinhole and goes through the pinhole and reaches the detector either PMT or CCD. Points above or below the focal plane cannot be imaged into the detect pinhole, the light emitted outside of the focal plane is blocked by the pinhole, it contributed very little to confocal images. Thus the confocal images substantially only obtain light information from the focal plane, namely acquired a 2D image sampled by the focal plane. If gradually adjusting the position of the longitudinal axis of the sample, multiple tomographic image of the sample can be generated and each crosssectional image of cell or tissue can be clearly displayed. This imaging method is called a confocal scanning microscope tomography. With three-dimensional image reconstruction technique, a high resolution three-dimensional image of the sample can be provided, as in many commercial instruments.
Fig. 2.1 Imaging principle of confocal laser scanning microscopy