High-definition whole-body vasculature imaging of live zebrafish larva obtained in a single FOV at frame rate of 500 Hz (see Table S1 for imaging parameters). (a) Color-coded maximum intensity projection (MIP) in the X Y plane over 425 μm along the Z direction. The 0 reference position of the MIP is marked by a red arrow in panel (b). (b) Color-coded MIP in the Y Z plane over 640 μm along the X direction indicated by the yellow dashed box in panel (a). The 0 reference position of the MIP is marked by a red in panel (c), (c) Color-coded MIP in X Z plane over 500 μm along the Y direction. The 0 reference position of the MIP is marked by a red arrow in panel (a). (d) Y Z cross sections of four pairs of ISVs indicted by corresponding double dashed lines in panel (c). (e)–(j) Enface z projections of the head region marked by yellow dashed box in panel (a). Paired vessel structures such as MCeV (middle cerebral vein), PHBC (primordial hindbrain channel), CCtA (cerebellar central artery), PMCtA (posterior mesencephalic central artery), AMCtA (anterior mesencephalic central artery), and PCS (posterior communicating segment) can be seen within different projections. Scale bar, 100 μm. (BCA, basal communicating artery; BA, basilar artery).

High-definition 3D imaging of an acute brain slice obtained in a single FOV at 3D cellular resolution across a wide FOV (see Table S1 for imaging parameters). (a) Color-coded MIPs of the enface view over a distance of 250 μm in the Z dimension, i.e., the whole brain slice volume. (b) Color-coded MIPs of the X Z plane over a distance of 450 μm in the Y dimension. The 0 reference positions of the projections are indicated by blue dashed lines in panel (a). (c),(e),(g) Zoomed images of the enface views of the areas indicated by dashed boxes in panel (a). (d),(f),(h) Color-coded MIPs of the Y Z plane of the volume indicated by double arrow lines in panels (c), (e), and (g). The scale bar for panel (a) is 200 μm; the scale bars for the other panels are all 100 μm.

Whole-body neuronal activity recording in zebrafish larva with 3D cellular resolution (see Table S1 for imaging parameters). (a) Color-coded MIP in X Y plane over 250 μm along the Z dimension. The reference positions of the MIP are marked by arrows in panel (b). (b) Color-coded MIP in X Z plane over 210 μm in the Y dimension. The reference positions of the MIP are marked by arrows in panel (a). (c)–(f) Enface projections at different z depths across the whole brain. (g)–(j) Zoomed views of the boxed areas in panels (c)–(f). (k) Representative neurons selected for evaluating correlated activity patterns are marked with circles; the color represents relative imaging depth from 0 to 120 μm. Fish data shown in (a)–(k) were acquired at 250 Hz per plane and scanned 125 planes at ~4 μm spacing, thus a 2 Hz whole-volume resolution. (l) Calcium traces of the selected neurons [see panel (l)] over an 85 s recording period. Scale bar, 100 μm.

Imaging blood flow over the whole larval zebrafish at high spatiotemporal resolution (see Table S1 for imaging parameters). (a) Single volume captured at volume rate of 1 Hz to highlight the structure. (b) Color-coded MIP of cell movements in the X Y plane. The X Y view is generated by spatial MIP along the Z direction over the position indicated by blue arrows in panel (c). (c) Color-coded MIP of cell movements in the X Z plane. The X Z view is generated by spatial MIP along the Y direction over the position indicated by blue arrows in panel (b). (d) Color-coded angiogram in X Y plane. (e)–(g) Tracking of blood cells in 3D over a time window of 4 s in a single ISV indicated in panel (e). Fish data shown in (b)–(g) were acquired at 625 Hz per plane and scanning 125 planes at ~4 μm spacing, thus a 5 Hz whole-volume resolution. (h) Measurement of velocities of blood cells in different types of vessels. The heart rate was estimated to be ~140 Hz by Fourier analysis. Scale bar, 100 μm.