Fluorescence-Based Imaging Techniques

Fluorescence imaging of reactive species (RS) using a variety of highly sensitive sensors allows temporal and spatial tracking of oxidants in uni- and multi-cellular organisms. Fluorescent probes are often oxidant sensitive, and they are non-fluorescent before being oxidized by RS. Upon reaction between probes and RS, the oxidized sensor becomes a highly fluorescent molecule. There is a wide range of chemical probes used in the biomedical field (e.g., dichlorodihydrofluorescein diacetate (DCFH-DA), dihydrorhodamine, dihydroethidium, Amplex Red, BODIPY, boronates, DanePy, DAF-2-DA, etc.) that exploit the existing oxidative chemistry to examine the production of RS in living cells. Genetically encoded biosensors based on fluorescent proteins (e.g. green fluorescent proteins (GFPs)), which provide a mean to real-time and dynamic detection of redox-status change without special requirements for permeability into target cells, are new and useful sensors to monitor changes in redox homeostasis during distinct physiological or pathological states. This chapter covers the up-to-date fluorescence imaging techniques for basic and clinical applications. It provides a concise, yet thorough, description of the state-of-the-art fluorescent probes that any investigator working in the field needs to access with their advantages and limitations. It also deals with nanodiagnostic methods for measuring oxidative stress (OS) with a special focus on the recent development in this field.