Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Area of Science:

• Biomedical Engineering
• Neuroscience
• Stem Cell Biology

Background:

• Human induced pluripotent stem cells (hiPSCs) are valuable for disease modeling, enabling studies from subcellular signaling to complex brain organoids.
• Advanced microscopy is crucial for imaging these 3D models, requiring a balance of optical sectioning, resolution, speed, and low invasiveness.

Purpose of the Study:

• To develop and characterize a novel microscope combining features of confocal and light-sheet microscopy for high-resolution 3D imaging of hiPSC-derived models.
• To overcome limitations of existing microscopy techniques for imaging complex biological structures like brain organoids.

Main Methods:

• A planar two-photon (2P) excitation microscope was engineered using a modified Nipkow-Petráň spinning-disk system with integrated micro-optics for virtual light-sheet generation.
• The system utilizes a single objective for both excitation scanning and fluorescence collection, coupled to a sCMOS camera for detection.
• Imaging was performed on 3D embryonic bodies (EBs) and brain organoids, both uncleared and partially cleared.

Main Results:

• The developed 2P-spinning disk microscope achieved high-resolution 3D imaging of hiPSC-derived tissues.
• Compared to confocal laser-scanning microscopy (CLSM), the new instrument demonstrated one order of magnitude faster imaging speeds.
• The microscope exhibited reduced photobleaching and enhanced depth penetration, crucial for thick biological samples.

Conclusions:

• The novel 2P-spinning disk microscope provides a powerful tool for advanced 3D imaging of hiPSC-based disease models.
• This technology enables more efficient and less damaging investigation of complex neural structures and developmental processes.
• The instrument's performance surpasses traditional CLSM for specific applications in stem cell research and neuroscience.