Imaging the Dynamics of Biological Processes via Fast Confocal Microscopy and Image Processing

Abstract

INTRODUCTIONConfocal microscopy offers the ability to optically section biological samples and to build three-dimensional (3D) volumes that can be digitally rendered, viewed from arbitrary directions, and quantitatively analyzed. Capturing, in addition to their 3D structure, the dynamics of living cells and organisms, further requires that image acquisition be performed rapidly to avoid measurements that are corrupted by sample movement. Several variations of the confocal principle have been incorporated into microscopes that can achieve fine optical sectioning, high frame rate, and high signal-to-noise ratio (SNR), while minimally perturbing biological samples. In this article we present some of the challenges and the requirements that are tied to imaging live samples and discuss how these are addressed by three optical versions of fast confocal microscopes: single-beam point scanning, spinning-disk, and slit-scanning confocal microscopes. Finally, we show that, for samples undergoing periodic motions, dynamic 3D image representations with virtually identical frame rates as in the case of two-dimensional (2D) imaging can be achieved via postacquisition image synchronization. These tools and techniques open new avenues for imaging fast dynamic processes in biology.