Optical section in embryonic spinal cord of zebrafish obtained with HiLo microscopy.

(A) Lateral view of the spinal cord of transgenic zebrafish embryo expressing the fluorescent protein GCaMP5 pan-neuronally in Tg(elavl3:GCaMP5G) obtained with HiLo microscopy. (B) Same image obtained in UWF mode. The spatial distribution of GCaMP5G remains hidden under a haze of background fluorescence due to missing optical sectioning.Scale bars in both panels are 20 μm. (C) Corresponding z-stack in 3D visualization, imaged in HiLo mode. The pools of neurons on each side of the spinal cord are visible in two separate planes. (D) Same reconstruction in WF mode (grid step 10 μm/line). See also S1 Movie.

HiLo microscopy enhances calcium signals.

(A) In vivo HiLo image of motor neurons of a zebrafish embryo expressing GCaMP5G in soma (ROI1), axons (ROIs 2–4), or background (ROIs 5–7). Scale bar 5 μm. (B) ΔF/F time series in the soma (ROI 1) shows that HiLo mode leads to a larger signal than UWF mode. (C) For axons (ROI 4) the gain in HiLo mode is even larger. Acquisition rate was 25fps.

Fast calcium imaging at 100 fps with HiLo microscopy reveals different dynamics in soma and initial segment.

(A) HiLo image of motor neurons expressing GCaMP5G with ROIs. Scale bar 5 μm. (B) Time course of calcium signals in soma, initial segment and axon, recorded at 100 fps. In the axon (ROI 1, gray) and the initial segments (ROI 4, light blue; ROI 5, magenta) a much faster rise is observed than in the somata (ROI 2, orange; ROI 3, dark blue). Somatic signals show a linear rise and a mono-exponential decay (Inset: blue somatic signal with red dotted fit, decay time 1.7 s, R² = 0.998). (C) The kinetics of calcium transients in different compartments were stable over time as shown with a recording performed 2.5 min later.