It is well established that a maleimide linker attached to a fluorophore acts as a fluorescence quencher. However, the maleimide double bond becomes saturated and fluorescence quenching is averted when maleimides react with thiols. To study the impact of protein conjugation via a thiol on the fluorophore performance, we coupled maleimide F1 and F2 with cysteine derivative 10 to give F5 and F6 (Scheme 3). Similar to compound F1, F5 showed nearly the same excitation and emission maxima values with a molar extinction coefficient value of 6169 L M -1 cm -1 (Table ). However, the quantum yield ΦF for F5 increased nearly four-fold (23.0%). Correspondingly, nitrogen derivative F6 showed similar excitation and emission maxima compared to its precursor F2. In this case, the quantum yield ΦF for compound F6 was even higher, increasing nearly six-fold (51.3%). To evaluate the value of these fluorescent small molecules within the context of in vitro and in vivo bioimaging of proteins, we established two different assays. In the first, we labelled the cytoskeletal protein actin, which assembles into polymers. Filaments composed of monomeric actins are readily visualised using conventional epifluorescence and/or total internal reflection microscopy. Purified actin was polymerised and then labelled with each of the compounds (F1-F4) through reaction of the native Cys 374 of actin with the maleimide-moiety of F1-F4. Actin bound to F1, F2, F3, and F4 was polymerised in vitro and imaged by spinning disk confocal microscopy by exciting the fluorophore with a 405 nm laser and collecting emissions using an EMCCD camera via the GFP emission filter which allows light of a wavelength of 505 nm and above to be detected. The polymers labelled with compounds F2 and F4 were readily detectable under our imaging conditions (Figure ). However, those labelled with compound F1 showed weaker fluorescence, and the fluorescence signal from filaments labelled with compound F3 was below detectable levels. The fluorescence intensity of filaments labelled with compounds F1, F2 and F4 were 1024, 2112, 1286 RFU respectively. As our in vitro experiments with labelled actin showed that compound F2 was the best suited in imaging proteins (and protein assemblies), we used it in further experiments to test its detectability in vivo. For these experiments we used zebrafish embryos for their ease of injectability and optical transparency which allows the introduction of the protein into the embryo and its live visualisation (Figure ). First, we injected embryos with F2 dye labelled actin protein and observed fluorescence at cellular margins likely to be cell junctions (Figure ). To exclude the possibility that the protein localisation was not merely an artefact of being labelled with the compound, we chose to label and observe a protein targeted to another cellular location, namely the nucleus. For this, we designed a construct that contained the SUMO tag for ease of purification, fused to two SV40 nuclear localisation signals (NLS) and three cysteines were engineered into this cysteine light NLS amino acid sequence for maleimide coupling. We purified and coupled this protein with compound F2 and injected it into embryos. The dye labelled SUMO-NLS protein showed clear staining in nuclear structures which were observed with differential interference contrast (DIC) imaging (Figure ). On the other hand, embryos injected with unlabelled SUMO-NLS protein showed very low autofluorescence which was not detectable at the same settings at which the dye labelled embryos were imaged (Figure ,f). Taken together, these results show that proteins can be successfully labelled with fluorophore F2. The labelled proteins retain their function and are detectable in vivo.