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RETINA Publication-derived

Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging

Source Browne et al., 2017 · USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of the University of Southern California · 10.1167/iovs.16-20796

👤 Andrew W. Browne, Cosimo Arnesano, Narine Harutyunyan, Thien Khuu, Juan Carlos Martinez, Harvey A. Pollack, David S. Koos, Thomas C. Lee, Scott E. Fraser, Rex A. Moats, Jennifer G. Aparicio, David Cobrinik ⏱ 151 days 📋 8 phases 🧫 Human ESC and Human iPSC

Abstract

This protocol characterizes hESC- and iPSC-derived retinal organoids using multimodal live imaging techniques including phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). The methods enable nondestructive assessment of retinal organoid structure and metabolic state during development into lamellar structures with photoreceptors.

Cell source
Human ESC and Human iPSC
Application
Developmental study

Protocol overview

20 steps across 8 phases

Stem Cell Culture and Organoid Differentiation 0-151 DIC
  1. 1 Culture hESC and iPSC and initiate differentiation into retinal organoids
  2. 2 Prepare organoids for live imaging
Phase Contrast Microscopy Concurrent with live imaging
  1. 1 Acquire phase contrast photomicrographs
Optical Coherence Tomography (OCT) Imaging Concurrent with live imaging
  1. 1 Suspend organoids in culture media
  2. 2 Perform OCT imaging with Spectralis system
Hyperspectral Imaging (HSpec) Analysis Concurrent with live imaging
  1. 1 Acquire spectral images with 2-photon microscopy
  2. 2 Perform phasor analysis of spectral data
  3. 3 Quantify biomolecule distribution by region of interest (ROI)
Fluorescence Lifetime Imaging Microscopy (FLIM) Analysis Concurrent with live imaging
  1. 1 Acquire fluorescence lifetime images
  2. 2 Generate phasor plot from FLIM data
  3. 3 Perform image segmentation and regional analysis
  4. 4 Quantify metabolic changes by free/bound NADH ratio
Histologic and Immunofluorescent Staining (Post-Live Imaging) After live imaging
  1. 1 Perform histologic staining
MicroCT Analysis of Fixed Tissue After live imaging or independent
  1. 1 Fix organoid tissue in formalin
  2. 2 Stain with Lugol iodine for contrast enhancement
  3. 3 Image with microCT immediately after staining
  4. 4 Perform high-resolution 3D scans
  5. 5 Reconstruct 3D images from CT data
Statistical Analysis After data collection
  1. 1 Analyze 2-photon light microscopy data
  2. 2 Calculate statistical significance

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Browne et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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