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In vivo and in situ measurements of osmotic pressure in blastomeres and in the interstitial fluid of zebrafish embryos a Confocal section of a Tg(actb2: mem-NeonGreen)hm37 zebrafish embryo transitioning from the 2- to 4-cell stage (membranes, yellow) with a droplet (fluorescent PEG in inner droplet, purple; fluorocarbon oil, cyan) located in one of the blastomeres (cells). b–b’, Close ups of the droplet in a. c Top panels: Confocal images of a droplet inside a cell of a developing zebrafish embryo at different developmental stages. Bottom panels: close ups of the droplet at each stage. d Representative example of measured time evolution of the intracellular osmotic pressure in a developing zebrafish embryo. N = 1. Mean ± measurement error bands (obtained by error propagation from calibration curve Fig. 2g) for d, f. e Timelapse of a zebrafish embryo in 2% w/w SDS solution imaged in an inverted microscope (transmitted light) and sustained on a porous membrane (Methods). f Representative time evolution of the osmotic pressure during SDS treatment (2% w/w SDS). N = 1. Insets show inner droplet equatorial confocal sections at different timepoints. g Confocal section of a zebrafish embryo blastula at sphere stage (4 hpf; same color code as in a) with a droplet inserted in the interstitial fluid between the cells. h Close up showing the equatorial confocal section of the droplet in g. i Schematic representation of the droplet in between adhering cells and the presence of osmolytes in the interstitial fluid. j Measured osmotic pressure inside blastomeres, between the cells (interstitial fluid) of the zebrafish blastula and after SDS treatment. N = 18, 20 and 10 droplets (1 droplet per embryo), respectively. Osmotic pressure of E3 buffer (embryo medium) with (violet line) and without (blue line) 2% w/w SDS, measured with a commercial osmometer (Methods). k Measured osmotic pressure variation (SD) of temporal readings in individual embryo (SD of temporal readings; N = 1) and across the different embryos (SD; N = 6). Boxplot show Median, 25th and 75th percentiles, whiskers extend to extreme data points. Source data are provided as a Source Data file.
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