As discovered by Abbé, a fundamental limit to the lateral resolution of a conventional optical microscope is produced by “far-field” aperture diffraction at the objective lens. This limitation can be expressed in terms of the Airy disc which relates the obtainable resolution to the wavelength of light used and the objective’s numerical aperture (NA). In scanning confocal microscopy with a laser-produced Gaussian profile illumination beam, the lateral resolution in given by 1.22λ/NA. The axial resolution in this design is improved by the use of a pinhole aperture to pass light only from the objective’s focal plane. Typical best values for lateral (x,y) and axial (z) resolution for visible wavelength scanning confocal microscopy are ~200nm and ~300nm with a NA=1.4 oil immersion objective.Although optical microscopy displays limited resolution when compared to some forms of electron and scanning-probe microscopies (STM, AFM), the convenience of sample preparation and its ease in performing in vivo studies has given rise to new methods of increased resolution (e.g., “near field” techniques, photon-tunneling, phase interference contrast).
Focal Surface Detection of High Numerical Aperture Objective Lens Based on Differential Astigmatic Method