Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Investigating cellular organelle spatial organization requires high-resolution 3D imaging.
• Current methods face limitations in resolving complex subcellular structures and molecular interactions.

Purpose of the Study:

• To develop and demonstrate a super-resolution microscopy technique for nanoscale 3D imaging of cellular components.
• To achieve high localization precision for visualizing intricate cellular architectures.

Main Methods:

• Utilized a 4Pi single-molecule switching super-resolution microscope.
• Employed a 'salvaged fluorescence' approach for ratiometric multicolor imaging.
• Achieved 5-10 nm localization precision in three dimensions.

Main Results:

• Successfully imaged the highly convoluted Golgi apparatus in mammalian cells.
• Resolved close contacts between the endoplasmic reticulum and plasma membrane.
• Demonstrated multicolor imaging of cellular structures at nanoscale resolution.

Conclusions:

• The 4Pi microscope with salvaged fluorescence enables unprecedented 3D nanoscale imaging of cellular ultrastructures.
• This technique bridges the gap between fluorescence microscopy and electron microscopy for cellular imaging.
• The salvaged fluorescence method is adaptable to various single-objective microscopes.