The Measurement of Free Calcium to Assess Cellular Function and Injury Using Digital Imaging Fluorescence Microscopy

Abstract

The purpose of this chapter is to introduce the technology involved in digital imaging fluorescence microscopy (DIFM) and its application to the study of toxic cell injury. A compelling issue in toxicologic pathology is the characterization of cellular and molecular events as they occur in the living cell following injury. The development of phase and Nomarski techniques for light microscopy has greatly improved the solution to this problem. Electron microscopy and analytical electron microscopy has enabled visualization of cell membranes and organelles at fixed points in time following an injury and has permitted correlation with observations of living cells in real time using phase or Nomarski microscopy often coupled with time-lapse cinematography or video microscopy. An important limitation of microscopy of living cells, however, has been that low levels of illumination and contrast problems have been difficult to solve and that information on chemical changes in the cell during phase microscopy has not been possible. Even the introduction of fluorescent probes has alone not solved these problems because of the phenomenon of photobleaching at normal levels of illumination. Moreover, the problems of contrast in examining the faint images of organelles were difficult to overcome even with advances in the sensitivity of photographic techniques. The development of DIFM has provided new opportunities for studying dynamic cellular events, particularly when coupled with traditional methods of fluorescence microscopy (for review see Taylor and Salmon, 1989).