Development of enhanced multi-spot structured illumination microscopy with fluorescence difference

Abstract

This project sets out to design and construct a novel super-resolution technique for biological imaging: enhanced multi-focal structured illumination microscopy; merging the fields of structured-illumination and point-spread function engineering. Computer simulations demonstrate the theoretical potential of this technique and suggest at least 1.4 times increase over existing structured illumination methods. Building on this, new pattern projection techniques based on holography are developed to project the required illumination patterns over an extended field of view. In addition, new techniques based on graphical processor unit programming are developed for the post-processing and reconstruction of multi-focal structured illumination data. Finally, these techniques are tested in the imaging of a range of biological structures in both living and fixed cells. While the holographic projection and post-processing techniques proved successful, the gains achieved with enhanced multi-focal structured illumination microscopy were limited. While there is a measured gain in resolution – and a potential improvement in depth sectioning – these advantages are not apparent on all structures. Finally, the relative merits of the techniques over existing methods are discussed and potential future directions are suggested.