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Example of multi color imaging of zebrafish eye using the custom built upright light sheet microscope

This movie shows division and migration of the retinal progenitors. Projections of Ptf1a inhibitory neurons (GFP+) can be seen as well.

Two transgenes were used: Ath5:gapRFP, where the Ath5 promotor drives the expression of membrane tagged RFP in retinal ganglion cells as well as in photoreceptors, horizontal and amacrine cells; and Ptf1a:cytGFP, where the Ptf1a promotor drives the expression of cytoplasmic GFP in amacrine and horizontal cells.

At the start of this movie, many of the cells are already labelled with RFP, whereas the GFP expression has just started in a few cells. As the retina develops, a wave of differentiation sweeps across the retina, as cells start to differentiate and migrate to their appropriate position. After 12 hours, the retinal ganglion cell layer is already established and we can observe some retinal ganglion cells axons exiting the eye and projecting towards the brain. At the same time, the amacrine cell layer has also been established. Towards the end of the movie we can see some Ptf1a+  horizontal cells migrating towards the edge of the retina, to establish the horizontal cell layer.

The imaging for both movies was performed on our custom built upright light sheet microscope, using sequential multi color excitation at 488 nm and 561 nm and acquiring volumes of  ~300umx300umx200um every 5 min. Shown are maximum intensity projections of the complete stack.

These images were acquired in collaboration with Xana Almeida, Harris group