100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales

Area of Science:

• Live-cell imaging
• Microscopy techniques
• Cellular dynamics

Background:

• Fluorescence microscopy is limited by photobleaching and motion blur, hindering dynamic studies.
• Investigating small objects requires high resolution and fast acquisition rates.
• Current methods struggle to capture rapid cellular events without artifacts.

Purpose of the Study:

• To introduce and validate an improved Total Internal Reflection-Rotating Coherent Scattering (TIR-ROCS) microscopy method.
• To demonstrate the capability of TIR-ROCS for high-contrast, label-free live-cell imaging.
• To analyze fast cellular dynamics and reveal hidden structural information.

Main Methods:

• Developed improved Total Internal Reflection-Rotating Coherent Scattering (TIR-ROCS) microscopy.
• Utilized variable illumination and intensity speckle pattern summation for high-speed imaging.
• Applied timescale decomposition and activity mapping for dynamic analysis.
• Achieved 160 nm resolution and 100 Hz imaging speed.

Main Results:

• Demonstrated high-contrast, label-free imaging of cellular dynamics at 160 nm resolution and 100 Hz.
• Visualized millisecond-scale actin cortex reorganization in macrophages.
• Captured fast degranulation and pore opening in mast cells.
• Revealed nanotube dynamics between cardiomyocytes and fibroblasts.
• Observed virus-sized particle binding and bacterial lectin dynamics.

Conclusions:

• TIR-ROCS microscopy overcomes limitations of traditional live-cell imaging techniques.
• The method allows for the study of rapid cellular processes previously obscured by motion blur.
• New analysis methods decipher how motion blur and slow motions mask critical fast cellular events.