eclipse ti2 e inverted spinning disc microscope  (Nikon)

Journal: The EMBO Journal

Article Title: MTCH2 controls energy demand and expenditure to fuel anabolism during adipogenesis

doi: 10.1038/s44318-024-00335-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Nikon ECLIPSE Ti2-E inverted microscope with a CSW-1 spinning disc system (Yokogawa) , Nikon , .

Techniques: Recombinant, Sequencing, Saline, Clinical Proteomics, Membrane, CyQUANT Assay, Proliferation Assay, Software, Western Blot, Reverse Transcription, SYBR Green Assay, Inverted Microscopy

Journal: Cell

Article Title: Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution

doi: 10.1016/j.cell.2020.08.013

Figure Lengend Snippet: Multilayered Human Retinal Organoids Produced in Quantity (A) Timeline of the AMASS organoid protocol with example bright-field images at important stages. Red arrowhead, pigment epithelium; red arrow, outer segments. EB, embryoid body. (B) Variability of embryoid body diameter with different generation methods. Bars, average coefficient of variation. Error bars, SD. (C) Embryoid body diameter (day 7) versus the number of cells seeded per microwell. Points, average diameter of embryoid bodies (n = 12) within an independent experiment. Line, quadratic fit. (D) Percentage of all organoids that were retinal organoids (week 6) versus embryoid body diameter (day 7). Points, experiments. Line, quadratic fit. (E) Schematic visualizing how AMASS improves yield from iPSCs using microwell array seeding and checkerboard scraping. (F) Comparison of organoid yield per well of iPSCs using different methods, weeks 20–38. Points, experiments. (G) Bright-field image of an organoid. OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (H) Confocal images. Left: adult retina. Right: organoid. Green, antibody against Bassoon (synaptic marker); white, Hoechst (nucleus marker). IPL, inner plexiform layer; GCL, ganglion cell layer. (I) Bright-field image of organoid pigment epithelial cells with black pigmentation. (J) Confocal image of pigment epithelial cells (maximum intensity projection). Magenta, MITF antibody; green, ZO-1 antibody (pigment epithelial cell markers). (K) Illustration of photoreceptor subcellular compartments. OS, outer segment; CC, connecting cilium; IS, inner segment; AT, axon terminal. (L–T) Organoid photoreceptors. (L–N) Outer segment. (L) Bright-field image. (M) Confocal images. Magenta, ARR3 antibody (cone marker); green, L/M opsin antibody (cone outer segment marker); white, Hoechst (nucleus marker). (N) Electron microscope image. Diagonal section of membrane discs. (O–Q) Connecting cilium. (O and P) Confocal images. Magenta, ARR3 antibody; green, ARL13B antibody (cilium marker). (P) Maximum intensity projection showing organoid surface. (Q) Electron microscope image. (R and S) Inner segment. (R) Confocal image. Magenta, L/M opsin antibody; green, TOMM1 antibody (mitochondrion marker). (S) Electron microscope image. (T) Axon terminal. Confocal image. Magenta, ARR3 antibody; green, RIBEYE antibody (ribbon synapse marker). All data from F49B7 organoids. See also Figure S1 , , and .

Article Snippet: Images were acquired using a spinning disc microscope (Nikon Ti2-E Eclipse inverted, Yokogawa CSU W1 dual camera T2 spinning disk confocal scanning unit, Visitron VS-Homogenizer).

Techniques: Produced, Comparison, Marker, Microscopy, Membrane

Journal: Cell

Article Title: Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution

doi: 10.1016/j.cell.2020.08.013

Figure Lengend Snippet: Characterization of F49B7 iPSCs and Retinal Organoids from Several iPSC Lines, Related to , , and and (A) Bright-field image of F49B7 iPSC colony. (B) Bright-field image of iPSC colony. Blue stain, alkaline phosphatase (pluripotency marker). (C – F) Confocal images of iPSCs. Green, antibody for pluripotency markers; white, Hoechst (nucleus marker). (C) SOX2, (D) NANOG, (E) OCT4 and (F) SSEA4. (G – I) Confocal images of iPSCs directly differentiated into the three germ layers. White, Hoechst. (G) Ectoderm; magenta, Nestin; green, PAX6 (ectoderm markers). (H) Endoderm; magenta, SOX17; green, FOXA2 (endoderm markers). (I) Mesoderm; magenta, NCAM; green, Brachyury (mesoderm markers). (J) G-banded karyotyping of F49B7 iPSCs. (K) Different iPSC lines generating 5-layered retinal organoids. Confocal images. Green, Bassoon antibody (synaptic marker); white, Hoechst (nucleus marker). Boxed area, outline of F49B7 image shown cropped in Figure 1 . (L) Retinal neuroepithelium (arrows) on a five-week old organoid. (M) Embryoid body diameter (day 7) versus the number of cells seeded per microwell. Points, mean diameter of embryoid bodies (n = 12) within an independent experiment. Line, quadratic fit. (N) Percentage of all organoids that were retinal organoids (week 6) versus embryoid body diameter (day 7). Points, experiments. Line, quadratic fit. (O) Confocal image. Green, RHO antibody (rod outer segment marker); white, Hoechst (nucleus marker). OS, outer segment; ONL, outer nuclear layer. (P – S) Electron microscope images of photoreceptor outer segment (OS; P, Q, S), inner segment (IS; P, R, S) and connecting cilium (CC; P, R, S). P and S are from serial sections. (T) Confocal image. Magenta, TRPM1 antibody (ON bipolar cell marker); green, GFP antibody (GCaMP6s); white, Hoechst (nucleus marker). (U) Scheme of the time course with which organoids were sampled for single-cell RNA sequencing. Data in A – J, K, L, O, P – S and T are from F49B7 organoids, data in K, M and N are from IMR90.4 organoids.

Article Snippet: Images were acquired using a spinning disc microscope (Nikon Ti2-E Eclipse inverted, Yokogawa CSU W1 dual camera T2 spinning disk confocal scanning unit, Visitron VS-Homogenizer).

Techniques: Staining, Marker, Microscopy, RNA Sequencing

Journal: Cell

Article Title: Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution

doi: 10.1016/j.cell.2020.08.013

Figure Lengend Snippet: Organoids Are Light-Responsive and Contain Functional Synapses (A–E) Synapses in organoids. (A, B, D, and E) Cone axon terminals. Confocal images. White, ARR3 (cone marker). Green, antibody against Bassoon (ribbon synapse marker); RIBEYE (ribbon synapse marker). Magenta, antibody against RIBEYE; PSD95 (postsynaptic marker); TRPM1 (ON bipolar cell marker); PV (horizontal cell marker). (C) Electron microscope image. AT, photoreceptor axon terminal; RS, ribbon synapse. (F) Schematic of organoid infection with adeno-associated viral vectors (AAV) expressing the calcium sensor GCaMP6s under the promotor EF1α. (G and H) Organoids expressing GCaMP6s. (G) Confocal image. Green, antibody against GFP detecting GCaMP6s. White, Hoechst (nucleus marker). C, cell with cone morphology; HC, cell with horizontal cell morphology; AC, cell with amacrine cell morphology; MC, Müller cell confirmed by counter-stain. (H) Two-photon microscope image. Optical cross-section of living organoid. Green, GCaMP6s. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (I) Organoid calcium activity during background illumination and light stimulation. Top, cells from ONL. Bottom, cells from INL/GCL. Black lines, peri-stimulus calcium activity (dF/F 0 ); line segments, individual trials. Red lines, scale bars. (J) Quantification of light responsive cells in ONL and INL/GCL. Error bars, 95% binomial confidence interval. (K and L) Organoid calcium activity during pharmacological block of glutamatergic synaptic transmission. CPP, (3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid); NBQX (2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline); APB, (2-amino-4-phosphonobutyric acid). (K) Responses from individual cells, as in (I). (L) Quantification of light responsive cells in ONL and INL/GCL, as in (J). All significance values from χ 2 test. All data from F49B7 organoids. See also Figure S1 .

Article Snippet: Images were acquired using a spinning disc microscope (Nikon Ti2-E Eclipse inverted, Yokogawa CSU W1 dual camera T2 spinning disk confocal scanning unit, Visitron VS-Homogenizer).

Techniques: Functional Assay, Marker, Microscopy, Infection, Expressing, Staining, Activity Assay, Blocking Assay, Transmission Assay