Simultaneous Two-Angle Axial Ratiometry for Fast Live and Long-Term Three-Dimensional Super-Resolution Fluorescence...

Area of Science:

• Biophysics
• Cell Biology
• Optical Imaging

Background:

• Four-dimensional (4D) super-resolution microscopy faces challenges with long acquisition times and high illumination intensity.
• Existing methods limit the speed and duration of live-cell imaging at the nanoscale.

Purpose of the Study:

• To introduce Simultaneous Two-Angle Axial Ratiometry (STARII) for enhanced axial super-resolution imaging.
• To enable fast and long-term imaging of live cellular processes with nanoscale resolution.

Main Methods:

• Utilizes evanescent wave illumination and records two raw images in two incident angle channels.
• Calculates the intensity ratio from the recorded images for axial localization.
• Combines STARII with lateral super-resolution techniques for 3D nanoscale imaging.

Main Results:

• Achieved axial super-resolution imaging with resolution below 20 nm.
• Enabled live-cell imaging at speeds up to hundreds of frames per second.
• Visualized nanoscale structures of microtubules, plasma membrane dynamics, and endoplasmic reticulum remodeling.

Conclusions:

• STARII overcomes key limitations in 4D super-resolution microscopy.
• The method facilitates investigation of complex cellular processes in their native state.
• STARII holds significant potential for advancing live-cell nanoscale imaging research.