Lightsheet z 1 microscope

Single Z-plane time lapses of 3 dpf zebrafish DA were acquired at 66 fps using a Zeiss Lightsheet Z.1 microscope. The resulting images were subjected to axial line scan particle image velocimetry designed for Lightsheet imaging using custom authored scripts in MATLAB 2017b®. Each time lapse was analysed frame by frame using intensity-based thresholding of circulating erythrocytes. Thresholded images were made binary followed by conversion of two-dimensional image to one-dimensional (1D) signal by summing up in ‘y’ direction (all column pixels). The 1D signal was low pass filtered to remove spurious and highly correlated pixels (low correlation implies displacement of 1D wave signal). Cross-correlation across time was calculated on filtered images for each time frame using inbuilt MATLAB 2017b® functions. Thus, the obtained correlation (in pixels/frames) was then converted to mm/s using the following equation: $$\mathrm{V}elocity\left(mm∕s\right)=\frac{Correlation\left(pixels∕frame\right)\times fps\times 10^{-3}}{Scale\left(pixels∕\mu m\right)}$$
Algorithm is available on request.

Images of immunostained samples were acquired using a Lightsheet Z.1 microscope (Zeiss) equipped with a 5× dry objective (NA 0.1) (Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, UNSW Sydney) using the following settings: Hoechst 33342: excitation 458 nm, emission bandpass 460–500 nm; Alexafluor 488: excitation 488 nm, emission bandpass 505–545 nm; DyLight 649: excitation 638 nm, emission long pass 600 nm. A tiled series of z-stacks between 800–1000 images with 10% overlap were acquired and processed with Vision 4D (Arivis) software.

Embryos were collected as above and stained as previously described (Wurdak et al., 2005 (link)). Embryos were mounted in low melting temperature agarose (Sigma-Aldrich, St. Louis, MO, USA). Prior to imaging, embryos were cleared in CUBIC reagent 2 for 24 h with gentle agitation. The embryos were imaged on a Zeiss Lightsheet Z.1 microscope with a 5× clearing objective. The imaging chamber was filled with CUBIC reagent 2 to match the refractive index of the cleared specimen.