Magnetic and Optical Molecular Imaging Live Cell and Tissue Fluorescence: Imaging Techniques and Methods

Abstract

Optical imaging of living cells provides a powerful suite of techniques for non-invasive linking of phenotypic expression at the biochemical level to individual genotypes. A plethora of fluorescent markers are available for selectively marking cellular components through monoclonal antibodies, specific ligand interactions, and through covalent bonding (Tsien and Waggoner 1995; Mason 2000). Imaging of fluorescent marker distribution within living cells has enabled measurement of many active physiological processes including protein transport, membrane potential, and free ion distributions (Terasaki and Dailey 1995). These markers, in conjunction with optical sectioning techniques such as confocal or multiphoton microscopy, allow interrogation of cellular dynamics to diffraction-limited spatial resolution even in thick tissues or cells. The main purpose of the first two sections of this chapter are to introduce the reader to various techniques and methods of fluorescence imaging of live cells and thick tissues. The descriptions are aimed at the interdisciplinary scientist unfamiliar with fluorescence imaging of thick (>5-10 μM) specimens. The fundamentals of optical sectioning through various forms of confocal microscopy and multiphoton microscopy will be addressed first, followed by a description of new or improved fluorescence techniques for dynamic live cell imaging. These descriptions will also emphasize the limitations and practical consideration of these techniques.