Home › Cell Biology › Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids
Cell Biology JoVE (Open Access) Citable · DOI

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

DOI: 10.3791/60709-v
What you'll learn
  • ✓ Perform single-cell resolution 3D imaging of intact organoids using confocal microscopy
  • ✓ Apply immunolabeling and optical clearing techniques to preserve organoid structure
  • ✓ Process and reconstruct 3D image data to visualize cellular architecture
Protocol

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Difficulty
advanced
Total time
~3–5 days (including fixation, immunolabeling, clearing, and image acquisition)

Steps

1
Recover organoids from culture

Extract organoids from growth medium and prepare for downstream processing. This step establishes the starting material for imaging.

▶ 00:46
2
Fix organoids and apply blocking

Chemically fix organoids to preserve morphology and block non-specific antibody binding. Essential for maintaining 3D structure during staining.

▶ 02:28
3
Perform immunolabeling on organoids

Apply fluorescent antibodies to detect cellular markers throughout the 3D organoid volume. Multi-day incubation optimizes penetration and signal.

▶ 03:30
4
Optically clear organoid tissue

Apply clearing reagents to reduce light scattering and improve penetration depth for confocal imaging. Enables visualization of interior structures.

▶ 05:33
5
Prepare samples for confocal imaging

Mount cleared organoids on imaging slides with appropriate mounting medium and coverslips. Ensures optical quality for microscopy.

▶ 06:08
6
Acquire and process 3D image stacks

Capture z-stack confocal images and apply computational processing to reconstruct 3D volumetric data. Generate single-cell resolution output.

▶ 07:01
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