Imaging single molecules using total internal reflection fluorescence microscopy (TIRFM).

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Total internal reflection fluorescence microscopy (TIRFM) offers superior signal-to-noise for visualizing fluorescent molecules near surfaces.
• It illuminates molecules within ~100 nm of the coverslip, enabling visualization beyond the diffraction limit (~200 nm).

Purpose of the Study:

• To review the theory, setup, and applications of TIRFM for biological research.
• To discuss advancements enabling single-molecule imaging at high concentrations and super-resolution localization.

Main Methods:

• Illuminating a thin volume near the coverslip using total internal reflection.
• Applying centroid-tracking methods for subdiffraction-limited localization precision (as low as 1 nm).
• Integrating advanced fluorophore technology and imaging techniques.

Main Results:

• TIRFM allows visualization of single molecules with high resolution and signal-to-noise ratio.
• Subdiffraction-limited localization precision of 1 nm is achievable with centroid-tracking.
• New techniques permit imaging of molecules at cellular concentrations and with super-resolution.

Conclusions:

• TIRFM is a powerful technique for studying protein dynamics, particularly near the plasma membrane.
• Recent advances have made TIRFM more accessible and versatile for biological imaging.
• The technique facilitates high-resolution imaging of single molecules in complex biological environments.