Fast 4D Microscopy

J.R. De Mey, P. Kessler, J. Dompierre, F.P. Cordelières, A. Dieterlen, J.‐L. Vonesch, J.‐B. Sibarita
Methods in Cell Biology. 2008-01-01; : 83-112
DOI: 10.1016/s0091-679x(08)85005-4

1. Methods Cell Biol. 2008;85:83-112. doi: 10.1016/S0091-679X(08)85005-4.

Fast 4D Microscopy.

De Mey JR(1), Kessler P, Dompierre J, Cordelières FP, Dieterlen A, Vonesch JL,
Sibarita JB.

Author information:
(1)Ecole Supérieure de Biotechnologie de Strasbourg, UMR-7175 CNRS/Université
Louis Pasteur (Strasbourg I), BP10413, 67412 IllKIRCH Cedex, France.

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.