Evolution of the number of GFP pixels based on GFP intensity thresholds for zebrafish embryos and regions of interest of fluorescent zebrafish applied with different thresholds. a Graphical representation of average GFP intensity thresholds on the x-axis and mean number of pixels greater than the threshold on the y-axis for the zebrafish embryos tested (n = 6). A progressive decay of the area, more evident at 72 hpi (dotted lines), is shown. It can also be observed that as the threshold increases, the area decreases slightly. At a low threshold, auto-fluorescence can represent an important component of GFP intensity. However, as soon as this threshold is raised, auto-fluorescence drastically disappears. Blue line represents 0 hpi embryos, and red line represent 72 hpi embryos. b Example of segmentation in evolution with red outlines over the images with thresholds from 0 to 50. The region inside the red outline is reduced as the threshold increases. This way the brightest pixels with higher fluorescence are selected, eliminating the majority of auto-fluorescence from the zebrafish embryo

Proliferation assay in zebrafish injected with different cell numbers at 34 °C and 36 °C. a, b Initial injected cells proliferation index at an incubation temperature of 34 °C (a: 100-200 cells, P.I. = 0.4603; b: 400-500 cells; P.I. = 0.7196). c, d Initial injected cells proliferation index at an incubation temperature of 36 °C (c: 100-200 cells, P.I. = 2.7558; d: 400-500 cells, P.I. = 1.9558). Images are representative of each of the conditions assayed. All images are a superposition of a fluorescence field image over a bright field image. In all panels the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm. P.I. = proliferation index. e Comparison between the initial number of cells injected (Low: 100-200 or High: 400-500) and their proliferation at two different temperatures tested (n_rep = 20-50, n_total = 207, ***p < 0.01)

Cell proliferation inside the zebrafish embryos at the two conditions tested. a Zebrafish embryo incubation at 34 °C, analyzed with ZFtool, yielded a proliferation index of 0.6253 (b) Zebrafish embryo incubation at 36 °C analyzed with ZFtool yielded a proliferation index of 2.4237. Images are representative of each of the conditions assayed. All images are a superposition of a fluorescence field image over a bright field image. In all panels, the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm. c Quantization of cell proliferation inside the embryos at the two temperatures tested in each experiment (34 °C-36 °C). (n_rep = 20-50, n_total = 207, ***p < 0.01)

ZFtool automatically elimination of fish autofluorescence. ZFtool software detects all the green pixels in the image (red/pink line) but eliminates all those pixels corresponding to fish autofluorescence and keeps pixels above an established threshold (blue line).

Automated counting of cells. This image shows the process of the software to count the cells on the microscope slide performed before the injection of the zebrafish embryos. (A) Fluorescent image of low cell number. (B) Cells of the A image counted (179). (C) Fluorescent image of high cell number. D: Cells of the C counted (404). Scale bar = 100 μm.

Cell proliferation inside the zebrafish embryos at 34 °C and 34 °C with 5-FU (A) Zebrafish embryo incubation at 34 °C analyzed with ZFtool yielding a proliferation index of 0.4748. (B) Zebrafish embryo incubation at 34 °C, with 5-FU analyzed with the ZFtool yielding a proliferation index of 0.5415. All images are a superposition of a fluorescence field image over a bright field image. In all panels, the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm.

Cell proliferation inside the zebrafish embryos at 36 °C and 36 °C with 5-FU (A) Zebrafish embryo incubation at 36 °C analyzed with ZFtool yielding a proliferation index of 2.6653. (B) Zebrafish embryo incubation at 36 °C, with 5-FU analyzed with the ZFtool yielding a proliferation index of 1.9592. All images are a superposition of a fluorescence field image over a bright field image. In all panels, the left image is a 48 hpf or 0 hpi zebrafish embryo, and the right image is the same zebrafish embryo with 120 hpf or 72 hpi. Scale bar = 100 μm.