Three-dimensional Imaging on Confocal and Wide-field Microscopes

Abstract

The conventional (wide-field) light microscope accepts light from planes above and below the plane of focus. This lack of depth discrimination is the main limitation of the wide-field microscope for 3D imaging. The confocal microscope rejects this out-of-focus light with the confocal pinhole and provides greater resolution than the wide-field microscope. This depth discrimination of the confocal microscope makes it attractive for 3D optical sectioning microscopy. This advantage of the confocal microscope is balanced by inherent signal losses from the confocal pinhole (see also Chapter 2: Pinhole) and by the use of detectors in current commercial confocal microscopes that have substantially lower quantum efficiency than the cooled CCD cameras used in wide-field digital imaging microscopes. These additional losses of current commercial confocal instruments are especially important in 3D fluorescence imaging of single cells.