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RETINA Publication-derived

Investigation of human organoid retina with digital holographic transmission matrix measurements

Source Koukourakis et al., 2022 · TU Dresden (Laboratory for Measurement and Sensorsystem Technique, Competence Center for Biomedical Laser Systems) · 10.37188/lam.2022.023

👤 Nektarios Koukourakis, Felix Wagner, Stefan Rothe, Mike O. Karl, Jürgen W. Czarske ⏱ 231 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human retinal organoids from induced pluripotent stem cells (iPSC) and their analysis using digital holographic transmission matrix measurements to distinguish healthy from CCCP-induced pathologic tissue. The transmission matrix fully describes light propagation through scattering retinal tissue, enabling detection of cellular changes and tissue degeneration.

Cell source
Human iPSC
Application
Disease modeling and optical characterization

Protocol overview

51 steps across 9 phases

hiPSC maintenance and preparation Days 0-5 (pre-organoid phase)
  1. 1 Maintain hiPSC culture
  2. 2 Dissociate and suspend hiPSC in Matrigel
  3. 3 Initiate floating culture in N2B27 medium
Early retinal organoid development and plating Days 5-30
  1. 1 Plate developing neuroepithelial cysts
  2. 2 Detach and transfer cell sheets to floating culture
  3. 3 Manually isolate retinal domains
  4. 4 Begin FBS supplementation and EC23 addition
Mid-stage organoid maturation Days 30-100
  1. 1 Maintain floating culture with B27+FBS medium
  2. 2 Perform half-medium changes every 2-3 days
  3. 3 Continue EC23 supplementation
Late-stage organoid maturation Days 100-220
  1. 1 Switch to N2+FBS medium at day 100
  2. 2 Discontinue EC23 supplementation after day 120
  3. 3 Maintain floating culture and half-medium changes
Disease model induction (optional pathology phase) Days 220-231 (11-day CCCP treatment)
  1. 1 Prepare mature organoids for treatment
  2. 2 Prepare CCCP solution
  3. 3 Apply daily CCCP treatment
  4. 4 Replace half medium every other day
  5. 5 Observe gross morphological changes
Sample preparation for transmission matrix measurements After day 231 or at desired endpoint
  1. 1 Fix organoids in 4% paraformaldehyde
  2. 2 Store fixed organoids in PBS
  3. 3 Prepare vibratome sections
  4. 4 Mount tissue sections on microscope slides
  5. 5 Position samples for transmission matrix measurement
Digital holographic transmission matrix measurement On demand (measurement phase)
  1. 1 Initialize single-mode laser at 532 nm
  2. 2 Configure beam path with polarization optics
  3. 3 Display phase masks on spatial light modulator
  4. 4 Relay SLM image and focus through microscope objective
  5. 5 Collect transmitted light through second objective
  6. 6 Record off-axis hologram on digital camera
  7. 7 Perform hologram reconstruction and Fourier filtering
  8. 8 Extract and normalize complex light-field data
  9. 9 Build transmission matrix column
  10. 10 Repeat for all focal positions
  11. 11 Store complete transmission matrix
Transmission matrix analysis and singular value decomposition Post-measurement (computational analysis)
  1. 1 Perform singular value decomposition on TM
  2. 2 Identify optimal truncation point
  3. 3 Truncate SVD to obtain signal singular values
  4. 4 Calculate characteristic metrics from SSV
  5. 5 Compare healthy vs. pathologic samples
  6. 6 Perform statistical analysis
  7. 7 Visualize and report findings
Immunohistochemistry and validation (optional companion analysis) Post-measurement (parallel validation)
  1. 1 Fix tissues for cryosectioning
  2. 2 Embed tissues in O.C.T. medium
  3. 3 Cryosection tissues at 12 μm thickness
  4. 4 Wash sections and apply blocking solution
  5. 5 Apply primary antibodies
  6. 6 Wash and apply secondary antibodies
  7. 7 Counterstain with DAPI
  8. 8 Perform TUNEL assay for cell death detection
  9. 9 Mount and image sections
  10. 10 Analyze and correlate with transmission matrix findings

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Koukourakis et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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