FIGURE SUMMARY
Title

Ultraspecific live imaging of the dynamics of zebrafish neutrophil granules by a histopermeable fluorogenic benzochalcone probe

Authors
Colucci-Guyon, E., Batista, A.S., Oliveira, S.D.S., Blaud, M., Bellettini, I.C., Marteyn, B.S., Leblanc, K., Herbomel, P., Duval, R.
Source
Full text @ Chem Sci
Fig. 5

HAB labels specific cells in live zebrafish from 32 hpf. Confocal fluorescence imaging of HAB labeling (10 μM) in live wild-type zebrafish embryos (32 and 48 hpf) and swimming larvae (72 hpf) following excitation at 488 nm and detection in the 550–650 nm range. The yellow-orange color is indicative of the fluorescence seen with the naked eye. Maximum intensity Z-projection images (2 μm serial optical sections) are shown. Arrows point to the HAB label; asterisks mark pigment cells. A, artery; N, notochord; V, vein.
Fig. 6

HAB labels zebrafish neutrophil granules. (a–l) Confocal fluorescence imaging of HAB (10 μM) in live transgenic 72 hpf zebrafish larvae following excitation at 448 nm under equilibrium conditions. Detection parameters were as follows: for HAB/mCherry: λex 448 nm, λem 550–650 nm, mCherry: λex 552 nm, λem 660–750 nm using sequential modes of acquisition. For GFP/HAB: GFP λex448 nm, λem 500–520 nm, HAB: λex 448 nm, λem 550–650 nm using sequential modes of acquisition. Maximum intensity Z-projection images (2 μm serial optical sections) are shown. (m–p) High-resolution DIC and confocal fluorescence imaging of HAB (10 μM) in live 72 hpf zebrafish larvae harbouring red neutrophils. Arrows point to HAB-labeled granules according to the DIC images in neutrophils. A single 0.4 μm optical section is shown. Boxes in (c), (g) and (k) indicate the regions magnified in the insets (d), (h) and (l) respectively. Abbreviations used: A (aorta); N (notochord); V (vein); asterisk = nucleus. See ESI for Videos S5 and S6† related to (m–p).
Fig. 7

HAB reveals the dynamics of neutrophil granules upon phagocytosis of zymosan particles in live zebrafish. (a and b) Confocal live imaging of HAB-labeled neutrophil granules upon phagocytosis of subcutaneously injected zymosan in a live 72 hpf zebrafish larva under diffusion conditions. HAB is recruited to the forming phagosomes (arrows). Inset: HAB labeling of a resting neutrophil. (c) Sudan Black (SB) staining of myeloperoxidase-containing neutrophil granules showing granule recruitment to the phagosome upon zymosan phagocytosis in fixed zebrafish larvae. Inset: SB staining of a resting neutrophil; a single 1 μm optical section is shown. (d) Frames extracted from an in vivo time-lapse confocal imaging sequence (time step = 1 min). Arrows point to HAB-labeled neutrophil granules that are recruited to the nascent zymosan containing phagosome. Three neutrophils (pointed with number 1 to 3) were tracked during the time lapse sequence. Maximum intensity Z-projection (1 μm serial optical sections). See ESI for Video S8† related to (d).
Fig. 8

HAB does not target zebrafish neutrophil myeloperoxidase, and its binding to neutrophil granules is not a general feature of chalcones. (a and c) Merged confocal fluorescence and bright-field imaging of HAB (10 μM) in live wild-type (a) or “Spotless” (NL 144_01 mutant: null mpx allele) (c) 72 hpf zebrafish larvae following excitation at 488 nm and detection in the 550–650 nm range under diffusion conditions. (b and d) SB staining of myeloperoxidase-containing neutrophil granules in bright-field imaging. (e–j) Merged confocal fluorescence and bright-field imaging of HAB (10 μM) in live wild-type 72 hpf zebrafish larvae co-treated with chalcones 15, 16 (flavokawain A), 17(cardamonin), 18 or 19 (100 μM) following excitation at 488 nm and detection in the 550–650 nm range under diffusion conditions. The yellow-orange color is indicative of the fluorescence seen with the naked eye. Single 2 μm optical sections are shown. Abbreviations used: A (aorta); N (notochord); UGO (urogenital opening); V (vein).
Fig. 9

HAB selectively labels live human primary neutrophils over other blood cell types. (a–c and d–f) confocal fluorescence and DIC imaging of HAB (10 μM) in live human neutrophils following excitation at 448 nm and detection in the 550–650 nm range. Single 0.4 μm optical sections are shown. (a–c) show resting, non-activated, non-adherent neutrophils. (d–f) show activated, adherent neutrophils: note HAB staining of neutrophils granules (g–i): confocal fluorescence and DIC imaging of HAB (10 μM) in live human lymphocytes (lc), erythrocytes (ec), monocytes (mc) and thrombocytes (tc) using the same settings as in (a–c) and (d–f). Single 0.4 μm optical sections are shown. The yellow-orange color is indicative of the fluorescence seen with the naked eye. Asterisk = nucleus.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Chem Sci
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