Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Confocal microscopy offers 3D precision but lacks speed and requires sample labeling.
• Multifocal imaging (MFI) provides high-speed 3D imaging but faces trade-offs between resolution, field-of-view, and label dependency.

Purpose of the Study:

• To develop an open-source 3D reconstruction algorithm for multifocal images.
• To enable fast, label-free 3D tracking of biological structures with high spatial and temporal resolution.

Main Methods:

• Developed an open-source 3D reconstruction algorithm for multifocal imaging data.
• Applied the algorithm to characterize fluid flow and flagellar beating in sperm cells.
• Utilized multifocal imaging (MFI) for high-speed, label-free 3D recordings.

Main Results:

• Achieved high-speed (500 Hz) 3D tracking with a z-precision of 0.15 µm in a large volume (240 × 260 × 21 µm).
• Successfully characterized fluid flow and flagellar beating of human and sea urchin sperm.
• Demonstrated label-free tracking of spherical and filamentous structures across large fields-of-view and depths.

Conclusions:

• The developed algorithm enhances multifocal imaging capabilities for studying fast biological processes.
• This cost-effective, label-free approach is broadly applicable in biophysics and cell biology.
• Enables simultaneous tracking of cellular trajectories and dynamic behaviors like flagellar beating.