Multiple spectral parameter imaging.

Publisher Summary This chapter discusses the requirements for performing multiple spectral parameter imaging on living cells. Many of these considerations also apply to fixed preparations. Cell functions, such as cell division, endocytosis, and cell migration involve a complex temporal and spatial interplay of multiple organelles, macromolecules, ions, and metabolites. Goal of cell biology is to define the role of each cellular constituent and to define the molecular interplay of multiple constituents that are required for the completion of specific cell functions. Therefore, a technique is required that will permit the quantitation of multiple physiological parameters in time and space within the same cells. Quantitative fluorescence microscopy, when combined with the increasing number of sensitive fluorescent and other luminescent probes available, offers a powerful approach for defining the chemical and molecular dynamics within living cells in time and space. An approach is developed that allows the analysis and correlation of up to five separate parameters based on the spectral isolation of distinct fluorescent probes. The use of a properly designed imaging workstation helps to employ these methods in both two and three dimensions and to harness the power of video-enhanced contrast microscopy.