Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal...

Area of Science:

• Cell biology
• Microscopy
• Biophysics

Background:

• Total internal reflection fluorescence microscopy (TIRFM) excels at visualizing cellular processes near the plasma membrane.
• TIRFM offers high sectioning capability without specific fluorescent probes but is limited to a single plane.

Purpose of the Study:

• To develop a computational method for reconstructing 3D images from TIRFM data.
• To achieve high axial resolution and temporal resolution for dynamic cellular events.

Main Methods:

• A versatile design enabling fast multiwavelength azimuthal averaging and incidence angle scanning.
• Computational reconstruction of 3D image sequences.
• Application to live adherent cells.

Main Results:

• Achieved 50-nm axial resolution over an 800-nm range above the coverslip.
• Obtained structural and dynamical information about 3D actin architectures.
• Deciphered Rab11a-dependent exocytosis events in 3D at seven stacks per second.

Conclusions:

• The developed method extends TIRFM capabilities to 3D imaging.
• Enables detailed structural and dynamic analysis of cellular processes in 3D.
• Provides new insights into membrane-associated events like exocytosis.