Improvement of axial resolution in confocal microscopy by use of heterodyne illumination

Abstract

Confocal microscopy has been widely used in many industrial and biological researches. There are many aspects to a confocal microscope that make it a much more versatile instrument than other microscopes. Generally the resolution of a confocal microscope is better than a conventional optical microscope by a factor of 1.4. Rejecting out-of-focus images by a pinhole, it can provide optically sectioned images. Unlike scanning electron microscope and other microscopes, it is fast, non-destructive and non-invasive method, so that it can provide 3D imaging sample such as live cells. Even though confocal microscope has better resolution than conventional optical microscope and optical sectioning capability, it is inherently limited by diffraction theory that the resolution determined by the wavelength of the light and the numerical aperture (NA) of the lenses. As a result of the diffraction the axial resolution is approximately three times poorer than its lateral counterpart. Several researchers have studied in order to improve axial resolution of confocal microscope. 4Pi confocal microscope, which makes a constructive interference of either the excitation wavefront in the common focus or the detection wave fronts in the common detector pinhole, archived an axial resolution approximately four times higher than that in an ordinary confocal microscope. Modifying the spatial arrangement of the excitation and detection point spread functions in a confocal arrangement or inserting pupil filter in the optical path improve their lateral and axial resolution. Nonlinear nature can be used to improve the resolution of confocal microscope. In this thesis, a new technique for improving the axial resolution of confocal microscope is proposed. This approach is based on the generation of a frequency domain field confined focal spot, which is made by heterodyne interference between two axially shifted spots. When two beams, which come from same sources but have different freq...