One revolution in the last decade has been the application of new imaging methods to modern biology, revealing new insights into cellular structure and dynamics. However, imaging methods capable of three-dimensional (3D) imaging at high spatial and temporal resolution are still lacking. This thesis describes two projects aimed at advancing volumetric whole-cell imaging based on fluorescence microscopy.

The first project establishes a robust single-molecule localisation microscopy (SMLM) pipeline for volumetric imaging in the whole cell using the double-helix point spread function (DH-PSF) approach. The practical challenges associated with this method, such as aberration- and drift-induced inaccuracies, were comprehensively evaluated, and methods were developed to address all these problems. With a novel spontaneous blinking PAINT (Point Accumulation for Imaging in Nanoscale Topography) dye, HMSiR-Hoechst, super resolved SMLM images of DNA throughout the entire mammalian cell nucleus were obtained.

The second project involved the design and construction of an epi-illumination selective plane illumination microscopy (eSPIM) system capable of rapid volumetric multi colour live fluorescence imaging of cellular samples. This system was one of the first of its kind in Europe, demonstrating excellent performance by yielding high resolution in line with established standards. A comprehensive comparison of eSPIM with three alternative volumetric imaging modalities was conducted, focusing on the dynamics of DNA and Heterochromatin Protein 1 beta (HP1β) within live mouse embryonic stem cells (mESCs), demonstrating the superior performance of the eSPIM.

Collectively, this work makes a significant contribution to volumetric fluorescence microscopy, extending our capability to dissect and visualise cellular structure and interaction in whole-cell in 3D.