Resonance Raman spectra of human macula.

<p>Room temperature spectra (900–1600 cm^-1) are shown for <i>ex vivo</i> human retina in the macular region, excited at 488.0, 501.7, 514.5 & 528.7 nm (blue, olive, black, red respectively). Details of the ν₁ & ν₄ regions are shown in the insets. Representative spectra are shown for a single macula, but were the same for all 8 subjects used in this study.</p

Resonance Raman spectra of MP carotenoids <i>in vitro</i>.

<p>Room temperature spectra in the 900–1650 cm^-1 region are shown for zeaxanthin (red) and lutein (blue) in THF, excited at 488.0 nm. Inset: detail of the ν₁ region.</p

Dynamic Full-Field Optical Coherence Tomography module adapted to commercial microscopes for longitudinal in vitro cell culture study

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organisation as well as cell shape, motility and division. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening

ν₁ region of macula resonance Raman spectra.

<p>77 K (solid lines) and room temperature (dashed lines) spectra are shown for excitation at 488.0 & 514.5 nm (blue, black respectively). For 488 nm, additional spectra are shown for measurements scanning in towards the foveal centre (dotted lines).</p

Survival of mixed neurons and glial cells on diamond.

<p>(<b>A</b>) protein-coated glass, (<b>B</b>) bare glass, (<b>C</b>) protein-coated H-terminated diamond, (<b>D</b>) bare H-terminated diamond, (<b>E</b>) protein-coated O-terminated diamond, (<b>F</b>) bare O-terminated diamond. Glial cells and bipolar cells were identified, with an anti-GFAP (green) and an anti-Goα (red) antibody, respectively. (G-H) Quantification of the neuronal bipolar cells (G) and glial cells (H: ×10³ μm²). (means ± SEM, <i>n</i> = 4 experiments, 3 samples/group/experiment). Two-way ANOVA was carried out, followed by a Bonferroni post-hoc test (**<i>p</i><0.01, *<i>p</i><0.05). The scale bar represents 50 μm.</p

Distinct glial and neuronal cell preference for diamond protein-patterned substrates.

<p>Pure postnatal (p7) retinal ganglion cells (RGCs) on a protein-patterned glass (<b>A</b>) or protein-patterned diamond (<b>B</b>) substrate. RGCs were identified on the basis of anti-NF200 immunolabeling (red fluorescence) on the protein pattern (green) (<b>C</b>). Mixed retinal cell culture showing the preference of the glial cells for the protein pattern on glass. (<b>D</b>) Quantification of the protein pattern surfaces on glass and diamond, with the overlap between RGCs and these protein patterns. (means ± SEM, <i>n</i> = 4 experiments). Two-way ANOVA was performed, followed by a Dunn's post-hoc test (***<i>p</i><0.001). The scale bar represents 50 μm.</p

Survival of pure adult retinal ganglion cell neurons on O-terminated diamond.

<p>(<b>A</b>) Protein-coated diamond, (<b>B</b>) bare diamond. (<b>C</b>) Quantification of viable retinal ganglion cells at 6 days <i>in vitro</i>, normalized with respect to the number of these cells on day 1 <i>in vitro</i>. (<b>D</b>) Quantification of neurite length at 6 days <i>in vitro</i> (means ± SEM from <i>n</i> = 4 experiments, 3 samples/group/experiment). Two-way ANOVA was performed, followed by a Bonferroni post-hoc test (***<i>p</i><0.001, *<i>p</i><0.05). The scale bar represents 50 μm.</p

Viability of mixed neurons and glial cells on diamond.

<p>(<b>A</b>) protein-coated glass, (<b>B</b>) bare glass, (<b>C</b>) protein-coated H-terminated diamond, (<b>D</b>) bare H-terminated diamond, (<b>E</b>) protein-coated O-terminated diamond, (<b>F</b>) bare O-terminated diamond. Note the presence of macroglial cells on the coated substrates (<b>A</b>, <b>C</b>, <b>E</b>) whereas small neuronal cells are visible on all substrates. The scale bar represents 50 μm.</p

Characterization of the poly-D-lysine coating on diamond.

<p>(<b>A</b>) Ellipsometry measurements on two H-terminated and two O-terminated diamond samples. Variation of apparent diamond thickness (nm) measured by ellipsometry after 45 minutes of incubation with water or poly-D-lysine (pDL). It seems to be possible to generate a measurable poly-D-lysine coating on hydrogen-terminated diamond surfaces, but not on oxygen-terminated diamond surfaces. (<b>B</b>) XPS spectrum on hydrogen-terminated diamond surface. N1s core level of poly-D-lysine coated H-terminated diamond. Data are expressed as means ± SEM from 3 experiments.</p

Increased TFTC Recruitment and Histone Acetylation at a Specific Subset of Deregulated Genes in R7E Retina

<div><p>ChIP assays were performed using formaldehyde-fixed chromatin extracts of retina from 2-mo-old control (WT or R7N; dark grey) and R7E (light grey) mice. Primers were selected to amplify promoter or enhancer regions of the specified genes, as depicted.</p> <p>(A and B) ChIPs using an antibody against a TFTC-specific subunit (Spt3) revealed an increased recruitment of Spt3 in R7E retina, at promoters from genes normally highly and specifically expressed in differentiated rods, namely<i>Rho, Pde6b,</i> and<i>Rbp3.</i> No such differences could be observed at promoters from two genes regulating rod terminal differentiation,<i>Crx</i> and<i>Nrl,</i> and at the intronic enhancer region of a house-keeping gene,<i>Ncl.</i></p> <p>(C and D) ChIPs using an antibody against acetylated lysines 9 and 14 of histone H3 (Ac H3) revealed an increased acetylation of histone H3 in R7E retina, specifically at the same promoters, which showed increased Spt3 binding<i>(Rho, Pde6b,</i> and<i>Rbp3)</i>. Promoters from<i>Crx, Nrl,</i> and<i>Ncl</i> did not show any differences in histone H3 acetylation.</p> <p>The amount of immunoprecipitated DNA was quantified by real-time PCR and normalized to the amount of DNA present in a fraction of the input chromatin extract. Values are expressed as fold enrichment over background IP signals obtained in corresponding no antibody ChIP experiments (see<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040067#sg009" target="_blank">Figure S9</a>). Each bar represents the mean value ± SEM (<i>n</i> = 4).</p></div