Journal of Histochemistry & Cytochemistry 59(2) 139 –145 © The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav

The goal of histochemistry is to provide color and contrast to microscopic images. The field uses disparate techniques to accomplish the specific labeling of biological structures. Histochemists pioneered the use of small-molecule cellular stains, labeled molecules such as antibodies, and enzyme-mediated detection and signal amplification (Kiernan 2008). Historically, however, histochemistry has been syn-onymous with the imaging of fixed (i.e., dead) cells and tissues. The advent of genetic manipulation techniques has greatly expanded histochemical methods to living cells. This review examines the current collection of labeling strategies and discusses the correlating fluorescent dyes that allow biologists to add color to live systems. Fluorescence and Fluorophores Fluorescence occurs when a dye absorbs one or more pho-tons, yielding a molecule in an excited state (Valeur 2002; Lakowicz 2006). This excited molecule can relax through a variety of processes, including the emission of a photon of another, typically longer, wavelength. It is this property— emission of a photon at a different wavelength—that allows detection of fluorophores in the presence of billions of other molecules in a biological sample. Thus, fluorescence-based techniques find widespread utility in bioresearch as they provide exquisite sensitivity and contrast in imaging exper-iments (Lichtman and Conchello 2005). Fluorescent molecules come in various forms, includ-ing small-molecule dyes accessed by organic chemistry (Haugland et al. 2005; Lavis and Raines 2008), the geneti-cally encoded fluorescent proteins (Giepmans et al. 2006; Chudakov et al. 2010), or the inorganic “quantum dots” (Smith A and Nie 2009). Here, we focus on the organic fluo-rophore classes, which encompass both synthetic small-molecule dyes and the fluorescent proteins. A key principle to understanding organic fluorophores is modularity. Building different substituents in and around the molecular structure of the dye allows tuning of the excitation maxima (λ ex) and emission maxima (λ em) across the entire visible spectrum and into both the ultraviolet and infrared (Lavi