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Fig. 3
Refractive index tuning with Iodixanol improves live-imaging of tissue culture systems.
(a). Effects of Iodixanol supplementation on live imaging of primary zebrafish cell cultures. Top Left: Brightfield image of a representative cluster of primary zebrafish embryonic cells, approximatly 50 µm in diameter. Centre panel: Images of cell clusters stained with the nuclear dye Hoechst 33342. Left column: 3D-reconstruction of representative multi-layered cell clusters, imaged in control media (RI = 1.333, top row) or in refractive index matched media (RI = 1.362, bottom row) under identical imaging conditions. The arrowheads indicate representative deep layer nuclei that are further shown as 2D optical XY-section in the right column. Graphs: Intensity profiles along the solid lines indicated in the respective xy-section image. The flatter and lower intensity profile in the control condition (top) quantitatively documents a loss of chromatin structure fine detail in deep nuclei, which is preserved by Iodixanol supplementation (bottom). Scale bars = 3D: 10 µm and 2D: 5 µm See Figure 3—figure supplement 1 for orthogonal sections. (b) Effects of Iodixanol supplementation on live imaging of human cerebral organoids. Top left: Dark field image of a representative human cerebral organoid approximately 2 mm in diameter. Centre panel: Human cerebral organoids at culture day 67 stained with the nuclear dye Hoechst 33342. Centre panel: 3D-imaging of organoids, mounted either in standard media (RI = 1.333, top row) or in refractive index matched media (RI = 1.363, bottom row) under identical imaging conditions. Left column: Maximum projections of representative z-stacks. The white frame indicates the region shown to the right as optical xy-sections at the indicated tissue depth. The solid white line across the deepest section traces the course of the pixel intensity profile shown to the right. The flatter and lower intensity profile in the standard condition (top) quantitatively documents the loss of nuclear signal at 40 µm depth, while Iodixanol supplementation (bottom) still allows nuclei detection at that depth. Scale bars = 50 µm. The color scheme encodes relative intensity (brightest = white).