2.1. General Principles of Fluorescence-Based Techniques

Fluorescence is the emission of light that occurs within nanoseconds after the absorption of light (i.e., excitation) that is typically of shorter wavelength [5]. The maximum emission wavelength is denoted as λem and the maximum excitation wavelength as λex. The λem and λex vary with different fluorescent molecules, which serve as the basis for detecting fluorescent substances. Fluorescence can be detected by fluorescence spectrophotometry (also known as fluorometry), visualized by fluorescence microscopy (i.e., fluorescence imaging), or perhaps more commonly by flow cytometry. Indeed, flow cytometry has become one of the most commonly used techniques in biomedical research.

Regardless of the instruments used, the techniques involve using a beam of light (ultraviolet or visible light) that excites the fluorescent molecules and causes them to emit light of a lower energy, typically, but not necessarily, visible light. For detecting ROS by fluorescence-based methods, a fluorescence-based detecting probe is incubated with the ROS-generating system (e.g., enzymes, cells, tissues), and the fluorescence of the reaction product is detected to determine the relative amounts of ROS. In general, most of the available fluorescence-based ROS-detecting probes are non-fluorescent or weakly fluorescent, but yield fluorescent products upon reacting with ROS.

Fluorescence-based probes permit ROS detection with much higher sensitivity than other techniques such as ultraviolet/visible (UV/Vis) spectrophotometry. In addition, fluorescence microscopy-based imaging allows in situ visualization of ROS formation in cells or tissues, and on the other hand, flow cytometry-based measurement provides dynamic information on cell populations. Limited selectivity for different reactive species is often a major disadvantage of the fluorescence-based techniques in detecting ROS production in biological systems. Nevertheless, when used appropriately and especially when combined with other assays, fluorescence-based flow cytometry can yield important information on the formation of ROS.