Flavin-based fluorescent proteins: emerging paradigms in biological imaging Arnab Mukherjee 1,4 and Charles M Schroeder 1,2,3 Flavin-based fluorescent proteins (FbFPs) are an emerging class of fluorescent reporters characterized by oxygen- independent fluorescence and a small size — key advantages compared to the green fluorescent protein (GFP). FbFPs are at a nascent stage of development. However, they have already been used as versatile reporters for studying anaerobic biosystems and viral assemblies. Recently, FbFPs with improved brightness and photostability have been engineered. In addition, several FbFPs show high degrees of thermal and pH stability. For these reasons, FbFPs hold strong promise to extend bioimaging to clinically and industrially significant systems that have been challenging to study using GFPs. In this review, we highlight recent developments in the FbFP toolbox and explore further improvements necessary to maximize the potential of FbFPs. Addresses 1 Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, USA 2 Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, USA 3 Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 4 Current address: Department of Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Corresponding author: Schroeder, Charles M (cms@illinois.edu) Current Opinion in Biotechnology 2015, 31:16–23 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes and Nicola Zamboni http://dx.doi.org/10.1016/j.copbio.2014.07.010 0958-1669/# 2014 Elsevier Ltd. All right reserved. Introduction Fluorescent proteins have revolutionized biological stu- dies by enabling imaging of molecular-scale events with high spatial and temporal resolution. In particular, the green fluorescent protein (GFP) and related analogs are widely used as genetically encoded reporters to investi- gate gene expression, protein localization, macromolecu- lar trafficking, and protein interactions [1,2]. However, despite more than a decade of discovery and engineering, all known GFP variants are strictly dependent on mol- ecular oxygen for maturation of fluorescence [3–7]. For these reasons, GFP-based proteins are dimly fluorescent to non-fluorescent in low-oxygen environments and have limited utility for studying anaerobic biosystems in- cluding high-density fermentations, cerebral ischemia, tumor hypoxia, pathogenesis, and biofilm development. Alternative fluorescent probes for imaging under low- oxygen conditions include small molecule organic dyes used in conjunction with biological labeling systems (e.g. SNAP-tags and HaloTags). However, cell-based fluores- cence imaging methods that rely on organic dyes can suffer from cytotoxicity, poor cell permeability, and high levels of background fluorescence arising from the need to remove excess unlabeled dye [8,9]. Consequently, the fluorescence reporter toolbox for low-oxygen imaging is severely inadequate for addressing a broad class of foun- dational and applied problems in anaerobic biology. From this perspective, there is a strong need for the develop- ment of new genetically encoded fluorescent proteins that are functional under anaerobic or low-oxygen con- ditions. Recently, a new class of flavin-based fluorescent proteins (FbFPs) derived from bacterial and plant photosensory flavoproteins has been developed and shown to fluoresce in an oxygen-independent manner [10  ,11  ,12]. In this way, FbFPs are promising candidates for addressing the long-standing challenge of developing viable genetically encoded fluorescent probes for imaging in low-oxygen conditions. In this review, we highlight key advances in the development and application of FbFPs and describe ongoing efforts to expand and diversify the FbFP imaging toolbox through protein engineering. In addition, we identify key challenges in FbFP-based imaging and suggest future directions to maximize the general utility and overall scope of FbFPs as an emerging class of fluorescent reporters. LOV-domain photoreceptors — molecular scaffolds for developing FbFPs FbFPs are derived from a highly conserved family of blue light photoreceptors known as light, oxygen, and voltage (LOV) sensing proteins. Wild type LOV proteins typi- cally associate with flavin mononucleotide (FMN) to function as light-driven regulators of diverse cellular functions ranging from stress response and virulence in microbes to phototaxis in plants and algae [13,14]. Upon blue light illumination, LOV proteins exhibit a complex photocycle that results in the formation of a covalent adduct between FMN and a cysteine residue located in the FMN-binding pocket. FMN-cysteine adduct Available online at www.sciencedirect.com ScienceDirect Current Opinion in Biotechnology 2015, 31:16–23 www.sciencedirect.com