Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Structured-illumination microscopy (SIM) offers super-resolution but often lacks speed and multicolor capabilities for live-cell imaging.
• Previous SIM implementations were too slow or single-color, limiting their utility for dynamic cellular processes.
• SIM's wide-field nature, light efficiency, and independence from field of view size make it ideal for live-cell imaging.

Purpose of the Study:

• To develop a novel SIM setup capable of fast, 3D, multicolor live-cell imaging.
• To overcome the speed and multicolor limitations of existing SIM techniques.
• To enable high-speed acquisition of dynamic biological events in living cells.

Main Methods:

• Developed a new SIM setup utilizing rapidly programmable liquid crystal devices.
• Implemented a flexible 2D grid pattern algorithm for rapid switching between excitation wavelengths.
• Achieved high volume acquisition rates for both single-color and two-color 3D datasets.

Main Results:

• Demonstrated a SIM setup capable of recording 3D two-color datasets of living whole cells at unprecedented speeds.
• Achieved volume rates of 4 seconds (one color) and 8.5 seconds (two colors) over multiple time points.
• Successfully imaged various cellular structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton.

Conclusions:

• The developed SIM microscope significantly advances live-cell imaging capabilities by enabling fast, multicolor super-resolution.
• This technology overcomes previous limitations, allowing for detailed observation of dynamic biological processes in 3D.
• The system is versatile and applicable to diverse biological samples, including cultured neurons and HeLa cells.