Confocal microscopy

General

Confocal microscopy (CM) is one of the most widely used high-resolution imaging techniques in tissue engineering (Smith et al., 2010). By introducing a spatial pinhole at the confocal plane in front of the detector, the out-of-focus light from the sample is eliminated, leading to a major improvement in axial resolution compared with a conventional wide field microscope (Gu, 1996). The spatial resolution of CM is mainly controlled by the diameter of the pinhole and is diffraction limited. Generally, with optimized conditions, the highest spatial resolution can reach ~0.8 pm in the axial direction and ~0.3 pm in the lateral direction. In comparison with traditional phase contrast microscopy, the confocal configuration of CM produces superior depthwise sectioning capability and is therefore able to provide high-resolution 3-D reconstruction. The imaging depth of CM is limited to the superficial layer of the sample (approximately 100-200 pm), which is dependent on the sample transparency and the wavelength of illumination. Current CM techniques largely rely on the scanning of the laser beam and have been extensively used for live imaging of cells and tissues, as well as structural characterization of scaffolds in tissue engineering (Hanke et al., 2003; Aigner et al., 1998; Engelmayr et al., 2008).

 
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