Fig. 4

Refractive index tuning with Iodixanol improves model organism live-imaging.

(a). Effects of Iodixanol supplementation on zebrafish embryo live imaging. Top left: Stereoscopic image of a dome stage zebrafish embryo of approximately 700 µm diameter. Centre panel: Zebrafish embryos expressing RFP-PCNA were injected at the single cell stage with 200 nm fluorescent sub-diffraction sized beads and imaged at dome stage (4 hpf). Images (left column: RFP-PCNA, right column: beads) represent 50 µm thick y-maximum projections of embryos imaged in regular media (RI = 1.333, top row) or in Iodixanol supplemented media (RI = 1.363, bottom row). Scale bars = 50 µm. The graphs to the right depict the quantification of point spread functions of individual beads (N = 20) at shallow (top) or deep (bottom) imaging depth, each for control and Iodixanol mounted specimens as per the indicated color scheme. The position of the analysed planes is indicated in the bead images to the left. The quantification of the width of the point spread function at half-maximal amplitude (See Supplementary file 1 for numerical results) reveals a significant increase in resolution in deep sections. (b) Effects of Iodixanol supplementation on planarian live imaging. Top left: Dark-field image of a specimen of the planarian flatworm Dendrocoelum lacteum approximately 4 mm in length. Centre panel: Live Dendrocoelum lacteum were stained with the nuclear marker RedDot1 and mounted in control media (RI = 1.333, top) or media Iodixanol-tuned to RI = 1.412 (bottom). The large images represent z-maximum projections of image stacks in the head region, with the solid line indicating the position of the single-plane orthogonal yz-section shown to the right. The scatter plots of mean nuclear intensity versus depth to the right quantitatively document an improved signal return upon Iodixanol supplementation, especially from deeper tissue layers. Scale bars = 50 µm. The color scheme encodes relative intensity (brightest = white).