A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Area of Science:

• Biophysics
• Optical Microscopy
• Molecular Imaging

Background:

• Structured illumination microscopy (SIM) is crucial for molecular-level visualization in sciences.
• Custom-built SIM systems can surpass commercial offerings in fidelity and speed.
• Existing systems often prioritize ease of use over advanced performance.

Purpose of the Study:

• To present a guide for constructing a high-performance SIM system.
• To achieve video-rate imaging (up to 10 Hz) with 100 nm resolution in three colors.
• To combine SIM with total internal reflection (TIR) for enhanced image contrast.

Main Methods:

• Utilized total internal reflection (TIR) illumination combined with SIM.
• Implemented automated control of illumination light's polarization and spatial structure.
• Ensured synchronization between illumination pattern, wavelength, polarization, and camera acquisition.
• Developed a step-by-step protocol for system alignment and calibration.

Main Results:

• Achieved 100 nm resolution imaging at up to 10 Hz in three colors.
• Demonstrated superior image contrast compared to other super-resolution techniques.
• Validated resolution improvement using standard test samples.
• Successfully performed video-rate super-resolution imaging on living cells.

Conclusions:

• The custom-built SIM-TIRF system offers significant advantages in speed and resolution.
• This system enables advanced visualization of dynamic molecular processes in living cells.
• The detailed guide facilitates the replication and advancement of high-performance microscopy techniques.