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Abstract

This chapter discusses the in vivo labeling of macromolecules and other cellular constituents and their measurement by fluorescence microscopy. An inherent pitfall in this approach is that either the labeling and/or observation technique may perturb the cell such that the information one derives is misleading or outright erroneous. The problem certainly applies to the determination of DNA content and cell cycle progression in living cells. The chapter describes the techniques for in vivo measurements; an implicit requirement is that all experiments must be carefully controlled in the sense of demonstrating that the conditions for labeling and measurement do not impair cell viability. In some cases, this condition cannot be met and there is no satisfactory alternative but to use techniques based on fixation and labeling protocols that help to take a snapshot in time of the DNA metabolism in the living cell. The chapter also presents the use of fluorescence digital imaging microscopy (FDIM) for measuring DNA, its metabolism, and its structure.