Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probeElectronic supplementary information (ESI) available: Detailed experimental procedures regarding compound synthesis, structural and spectral characterization, pKa prediction, bioimaging experiments, staining dynamics of HAB in 72 hpf zebrafish larvae (Videos) and complementary figures. See DOI: 10.1039/c8sc05593a

• Main Authors: Colucci-Guyon, Emma, Batista, Ariane S, Oliveira, Suellen D. S, Blaud, Magali, Bellettini, Ismael C, Marteyn, Benoit S, Leblanc, Karine, Herbomel, Philippe, Duval, Romain
• Format: Article
• Language: English
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/c8sc05593a

Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe ( HAB ) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB -stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB 's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems. A fluorogenic benzochalcone specifically labels live neutrophil granules in whole wild-type, GFP- or RFP-expressing zebrafish embryos and larvae.