Fig. 5

Selective imaging of nirFAST in chicken embryo and zebrafish larvae.a?c Plasmids encoding nirFAST-P2A-EGFP and frFAST-P2A-EGFP were electroporated in each side of the neural tube in ovo at embryonic day 2. 24?h later, embryos with homogenous bilateral reporter expression in the neural tube were dissected and imaged upon the addition of 10?µM of HPAR-3OM or HPAR-3,5DOM by timelapse spinning disk microscopy. Scale bars 100?µm. Representative timelapse micrographs of n?=?3 independent experiments. d Plasmids encoding nirFAST-P2A-EGFP and emiRFP670-P2A-EGFP were electroporated in each side of the neural tube in ovo at embryonic day 2. Twenty-four hours later, embryos with homogenous bilateral reporter expression in the neural tube were dissected and the neuroepithelium was imaged in en-face view by timelapse spinning disk microscopy upon sequential addition of 0.2, 1, and 10??M HPAR-3,5DOM. Scale bars 100?µm. Representative micrographs of n?=?3 independent experiments. e Plasmids encoding H2B-pFAST (cyan), memb-mCherry (yellow) and mito-nirFAST (magenta) were electroporated in the neural tube in ovo, at embryonic day 2 (E2, HH stage 13?14). Twenty-four hours later, embryos were dissected, and the neuroepithelium was imaged in en-face view in the presence of 1??M HMBR (to assemble pFAST540) and 10??M HPAR?3,5DOM (to assemble nirFAST715) using a spinning disk microscope (see also Supplementary Movie 2). Scale bars, 10?µm. Representative micrographs of n?=?3 independent experiments. f, g Mammalian HEK293T were transfected with plasmid encoding nirFAST-P2A-EGFP. After 24?h, they were injected near the heart of 2 dpf zebrafish larvae. Larvae with green fluorescence signal were selected and imaged at 4 dpf after 45?min incubation with HPAR-3,5DOM (to assemble nirFAST715). Representative micrographs of n?=?3 independent experiments. Scale bars, 20?µm. See Supplementary Table 8 for detailed imaging settings.