ABSTRACT
Autouorescence from cells has been detected and utilized by scientists for well over 100 years for a variety of applications. e need for uorescence as a noninvasive tool for cell studies was spurred by advances in optical microscopy, fueling the need for better contrasting agents to image samples. Initially, uorescent dyes such as uorescein, acridine orange, and neutral red provided a very high level of contrast due to their specicity in attaching to specic proteins in plant and animal cells. Cell and tissue autouorescence was simply observed as a biological phenomenon that tended to interfere with the signal and reduce image contrast. It was not until the landmark studies of Britton Chance (Chance and Williams 1955a,b,c; Chance et al. 1962) that autouorescence from cells was attributed to specic coenzymes, such as pyridine nucleotides, or avoproteins in mitochondria. Further, it was argued that because the oxidized pyridine nucleotides (NAD+) and the reduced form of avins (FADH2) were not uorescent, it was possible to ascertain the reduction-oxidation (redox) state of cells. Since then, autouorescence from cells in the form of NADH and FAD uorescence has been used in many studies to investigate the metabolic activities of various organs. Specically, some of the very rst in vivo studies for examining intracellular redox states were performed using singlephoton uorescence microscopes (Chance et al. 1962). In this chapter, we discuss the important components that are essential in the design of uorescence microscopy, calibration techniques, and image processing methods.
