Single molecule imaging in live zebrafish embryos by RLSM. a Sketch of reflected light-sheet microscopy of a zebrafish embryo. Inset: close-up view of a single cell within the embryo. b Sketch of mEos2-TBP and mEos2-Sox19b fusion proteins. mRNA coding for the constructs was injected into the 1-cell stage embryo. c Western blot analysis of TBP in wild type (wt) embryos (left lane) and injected with mEos2-TBP (right lane). ?-tubulin was used as loading control. Expected sizes are 50 kDa (?-tubulin), 33 kDa (wt-TBP) and 59.4 kDa (mEos2-TBP). d ChIP-qPCR analysis of 2xHA-mEos2-TBP (blue, 3 technical replicates) binding to chromatin in the promoter region of genes hmga1a, brd2a and a genomic control and corresponding IgG controls. Values are given in % of input (mean?±?s.e.m). e ChIP-qPCR analysis of 2xHA-mEos2-Sox19b (red, 3 technical replicates) binding to chromatin in the promoter region of genes dusp6, apoeb, pcdh18a and a genomic control and corresponding IgG controls. Values are given in % of input (mean?±?s.e.m.). f Effect of mEos2-TBP and mEos2-Sox19b expression in embryos grown at 22?°C (up to 6-somite stage) or at 25?°C until 24 hpf. The scale bar is 100 µm up to the 6-somite stage and 1 mm at 24 hpf. g Fluorescence images of a 64-cell stage embryo and an oblong stage embryo expressing mEos2-TBP. The surface of the animal cap and the outline of the nucleus are indicated (white lines). Red arrows point to single mEos2-TBP molecules. The scale bar is 10 µm. h Time traces of mEos2-fluorescence of the molecules indicated in g. i Signal-to-noise ratio of single nuclear mEos2-TBP molecules as a function of nucleus distance from the surface of the animal cap. Values are calculated from >200 detected molecules within each nucleus (data from 3 embryos, mean?±?s.e.m.)