Fluorescence microscopy offers unrivalled insight into the inner workings of dynamic biological processes across many temporal and spatial scales, with innovations such as light-sheet and confocal microscopy extending this imaging ability into all three spatial dimensions. When performed with these methods, however, 3D imaging lacks the high temporal resolution of 2D imaging. This is because acquisition of a volume requires the sequential acquisition of many individual 2D planes, with volumetric acquisition times often spanning multiple seconds. This means that such methods are unsuited to the 3D imaging of dynamic processes that occur over shorter timescales.

Ideally, it would be possible to perform full 3D imaging in a single snapshot, in a similar fashion to 2D imaging. Yet, while snapshot volume-imaging methods do exist, they typically require a significant compromise in spatial resolution. We see, therefore, that with existing methods there is a trade-off: for 3D fluorescence imaging we must choose between techniques that provide high-resolution in space, but slow, plane-by足plane 3D imaging; or those that provide fast 3D imaging but at the cost of spatial resolution.

In this thesis we introduce imaging techniques that overcome these limitations. Through the development of an image processing pipeline that offers volumetric re足construction from 2D projection images, we demonstrate how 3D fluorescence imaging may be performed while maintaining high resolution in both time and space. We apply the volume reconstruction framework to both simulated and experimental images ac足quired via two distinct projection imaging modalities, demonstrating the versatility of the technique. Additionally, we explore the benefits provided by the volumetric recon足struction pipeline in the context of single-molecule localisation microscopy. Because of the high temporal resolution offered by the volumetric imaging techniques introduced in this thesis, the methods are applicable to a wide range of dynamic biological samples and have the potential to offer new insight into dynamic biological processes. To the best of the author’s knowledge, the snapshot volume imaging methods introduced here are the first such methods that maintain the resolution of the parent imaging system.