Micro-Rotation Imaging- a novel system and new mathematical methodologies for three-dimensional fluorescence imaging in individual, non-adherent living cells.

Quantitative three-dimensional (3D) imaging of single living cells comprises an important tool for studying cell biology, cellular signalling and processes of infection and virulence therein. To date, conventional 3D imaging is limited mainly to methods using z-stack axial sampling whereby a sample volume is imaged repeatedly while mechanically shifting the microscope objective focal plane in small (100-500nm) axial steps. The resulting planar axial "z-stack" image series can then be processed and rendered in 3D using computer-software. However, among the main disadvantages of "through-stack" 3D imaging methods is the prerequisite that targeted samples must be stabilised by attachment to an optically transparent surface, precluding 3D visualization of many important non-adherent cell types, for example lymphocytes. Driven by the need to overcome this limitation we present a new micro-manipulation device (the Micro-Rotator*) that uses dielectric field control, enabling individual, non-adherent cells to be immobilised in suspension, and rotated around a fixed focal plane, through any chosen vector including the z-axis. We show that image series acquired during these z-axis rotation protocols effectively comprise fixed viewpoint 3D "movies" of compartmentalized fluorescence localised to single, living cells. Inasmuch as this method does not require axial movement of microscope or image detector components, and uses x,y plane sampling in one focal plane only, it offers a two- to three-fold superior 3D spatial resolution, and greatly diminishes (or removes completely) artefacts due to axial optical defects. As such, micro-rotational 3D imaging presents a distinctly new technological advance over existing axial through-stack methods, and opens new avenues requiring development of novel mathematical approaches to solve the reconstruction problem.

Work funded by EU-FP6-NEST programme in a grant to SLS, in consortium AUTOMATION (www.pfid.org/AUTOMATION)

*Patent: Shorte, S.L., Müller, T., Schnell, T. "Method and device for 3 dimensional imaging of suspended micro-objects providing high-resolution microscopy" Patent Number; EP 02 292 658.8; 25 October 2002