One-Photon Autofluorescence Microscopy
Autofluorescence from cells has been detected and utilized by scientists for well over 100 years for a variety of applications. The need for fluorescence 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, fluorescent dyes such as fluorescein, acridine orange, and neutral red provided a very high level of contrast due to their specificity in attaching to specific proteins in plant and animal cells. Cell and tissue autofluorescence 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 autofluorescence from cells was attributed to specific coenzymes, such as pyridine nucleotides, or flavoproteins in mitochondria. Further, it was argued that because the oxidized pyridine nucleotides (NAD+) and the reduced form of flavins (FADH2) were not fluorescent, it was possible to ascertain the reduction-oxidation (redox) state of cells. Since then, autofluorescence from cells in the form of NADH and FAD fluorescence has been used in many studies to investigate the metabolic activities of various organs. Specifically, some of the very first in vivo studies for examining intracellular redox states were performed using single-photon fluorescence microscopes (Chance et al. 1962). In this chapter, we discuss the important components that are essential in the design of fluorescence microscopy, calibration techniques, and image processing methods.