Fast 4D Microscopy.

Many cellular processes involve fast movements of weakly labeled cellular structures in all directions, which should be recorded in 3D time-lapse microscopy (4D microscopy). This chapter introduces fast 4D imaging, which is used for sampling the cell's volume by collecting focal planes in time-lapse mode as rapidly as possible, without perturbing the sample by strong illumination. The final images should contain sufficient contrast allowing for the isolation of structures of interest by segmentation and the analysis of their intracellular movements by tracking. Because they are the most sensitive, systems using wide-field microscopy and deconvolution techniques are discussed in greater depth. We discuss important points to consider, including system components and multifunctionality, spatial resolution and sampling conditions, and mechanical and optical stability and how to test for it. We consider image formation using high numerical aperture optics and discuss the influence of optical blur and noise on image formation of living cells. Spherical aberrations, their consequences for axial image quality, and their impact on the success of deconvolution of low intensity image stacks are explained in detail. Simple protocols for acquiring and treating point spread functions (PSFs) and live cells are provided. A compromise for counteracting spherical aberration involving the use of a kit of immersion oils for PSF and cell acquisition is illustrated. Recommendations for evaluating acquisition conditions and deconvolution parameters are given. Finally, we discuss future developments based on the use of adaptive optics which will push back many of today's limits.