6] - Techniques and Technology for Dynamic Video Imaging of Cellular Fluorescence

Abstract

Dynamic video imaging of cellular fluorescence, in conjunction with the wide variety of sensitive fluorescent probes now available, offers a powerful technique for investigating complex chemical and molecular mechanisms in single living cells. This technique, which is also called quantitative fluorescence microscopy, has evolved out of the development and applications of low-light-level cameras to detect faint fluorescence signals coupled with video-enhanced contrast microscopy. Fast computer-controlled microelectronic circuitry, which can be interfaced to these low-light-level cameras, has been developed so that video images can be rapidly captured, averaged, and stored in digital format in real time for later processing. Thus, dynamic video imaging microscopy allows acquisition of multiple images with different sets of excitation and emission wavelengths over a relatively short period of time. Use of dynamic video imaging microscopy with fluorescence probes is a powerful analytical tool for a variety of reasons. This chapter highlights these reasons. It also discusses various aspects of digital video imaging and presents a review of some of the fluorescent probes used and of the various aspects of the software and hardware required to collect and analyze the data. These points are illustrated with several examples of data obtained with a newly developed system.