Investigations in real-time confocal microscopy

Abstract

This thesis is a study of imaging in real-time confocal imaging systems. The particular instrument examined is the direct-view microscope (DVM), an instrument which allows real-time confocal imaging through the use of an array of pinholes rather than the usual single pinhole. The emphasis is placed on the depth sectioning side of the imaging: the greatest advantage offered by confocal systems, and also on the use of coherent illumination sources in such systems. We begin by introducing the elementary theory underlying all equations used in the thesis. The theory is both scalar and paraxial and describes the imaging accurately in systems of low angular aperture. The operation of the DVM in fluorescence mode is first studied. We experimentally examine the predictions of the scalar diffraction theory with respect to source coherence in such systems. Predictions of the theory are verified by employing a further paraxial modification of the usual equations to account for the non-ideal nature of our experimental setup. We make some broad comments about the general trade-offs between coherent and incoherent sources in fluorescence direct-view microscopy.