A short guide on blue fluorescent proteins: limits and perspectives

The advent of the so-called colorful biology era is in line with the discovery of fluorescent proteins (FPs), which can be widely used to detect the intracellular locations of macromolecules or to determine the abundance of metabolites in organelles. The application of multiple FPs that emit different spectra and colors could be implemented to precisely evaluate cellular events. FPs were initially established with the emergence of the green fluorescent protein (GFP) from jellyfish. Red fluorescent proteins (RFPs) from marine anemones and several corals adopt fluorescent chromophores that are similar to GFP. Chromophores of GFP and GFP-like FPs are formed through the oxidative rearrangement of three chromophore-forming residues, thereby limiting their application to only oxidative environments. Alternatively, some proteins can be fluorescent upon their interaction with cellular prosthetic cofactors and, thus, work in aerobic and anaerobic conditions. The modification of an NADPH-dependent blue fluorescent protein (BFP) also expanded its application to the quantization of NADPH in the cellular environment. However, cofactor-dependent BFPs have an intrinsic weakness of poor photostability with a high fluorescent background. This review explores GFP-derived and NADPH-dependent BFPs with a focus on NADPH-dependent BFPs, which might be technically feasible in the near future upon coupling with two-photon fluorescence microscopy or nucleic acid-mimickers. KEY POINTS: • Oxidation-dependent GFP-like BFPs and redox-free NADPH-dependent BFPs • GFPs of weak photostability and intensity with a high fluorescent background • Real-time imaging using mBFP under two-photon fluorescence microscopy.