Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy

Area of Science:

• Microscopy
• Biophysics
• Cell Biology

Background:

• Four-dimensional (4D) imaging demands high spatiotemporal resolution, speed, and minimal photobleaching.
• Existing methods like spinning-disk confocal and Bessel beam microscopy have limitations in resolution, speed, or phototoxicity.

Purpose of the Study:

• To develop a microscopy technique with enhanced spatiotemporal resolution and reduced photobleaching for 4D imaging.
• To enable high-speed, long-term volumetric imaging of dynamic biological processes.

Main Methods:

• A dual-view plane illumination microscope was engineered, alternating illumination and detection between perpendicular objectives.
• Computational fusion of dual-view data achieved isotropic 330 nm resolution.
• Achieved imaging speeds of 200 images/s, capturing 50-plane volumes in 0.5 seconds.

Main Results:

• Demonstrated isotropic resolution of 330 nm.
• Attained imaging speeds of 200 images/s with negligible photobleaching over hundreds of volumes.
• Successfully applied the technique to track microtubules in live cells, image nuclear dynamics during nematode embryogenesis, and visualize neural development in C. elegans.

Conclusions:

• The dual-view plane illumination microscope offers superior spatiotemporal resolution and speed with minimal photobleaching.
• This technique is well-suited for studying dynamic biological processes requiring extended high-speed volumetric imaging.