Spinning disk-remote focusing microscopy

Area of Science:

• Biophysics
• Microscopy
• Neuroscience

Background:

• Confocal microscopy is crucial for live-sample imaging but often limited by speed.
• Achieving high resolution over large axial ranges remains a challenge for optical sectioning techniques.

Purpose of the Study:

• To develop a fast confocal imaging method for high-resolution live-sample visualization.
• To enable the study of dynamic biological processes at the cellular level.

Main Methods:

• Combined remote focusing with differential spinning disk optical sectioning.
• Utilized a water-index calibration slide for precise alignment and minimized image distortion.
• Achieved axial and lateral resolutions of <5 µm and 490 nm, respectively, over a 130 µm axial range.

Main Results:

• Demonstrated a 256 × 128 µm field of view with minimal image volume distortion.
• Acquired image volumes at 1 volume/s over a 24 µm axial range.
• Successfully detected calcium-based neuronal activity in *Platynereis dumerilii* larvae.

Conclusions:

• The developed fast confocal imaging technique provides high-resolution 3D visualization of live biological samples.
• This method facilitates the real-time observation of cellular dynamics, such as neuronal activity.
• Offers a powerful tool for advancing live-cell imaging and neuroscience research.