Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.

Area of Science:

• Cell Biology
• Biophysics
• Optical Microscopy

Background:

• Single-molecule superresolution imaging is vital for cell biology.
• Limited depth of field restricts 3D imaging of whole cells.
• Existing methods (PALM, STORM) have shallow effective depths (<1.2 µm), causing artifacts and data loss.

Purpose of the Study:

• To develop a method for volumetric multicolor superresolution imaging.
• To overcome the depth limitations of current 3D superresolution techniques.
• To enable rapid imaging of nanoscale morphology in whole cells.

Main Methods:

• Utilized multifocus microscopy to capture nine focal planes simultaneously.
• Applied to single-molecule localization microscopy (SMLM) with fluorescent proteins and synthetic dyes.
• Achieved simultaneous volumetric imaging over an ~4-µm depth.

Main Results:

• Demonstrated lateral and axial localization precisions of ~20 nm and ~50 nm, respectively.
• Enabled instantaneous capture of molecular distributions within the ~4-µm volume.
• Successfully imaged the 3D organization of mammalian mitochondrial networks and yeast microtubules.

Conclusions:

• Multifocus microscopy significantly advances 3D superresolution imaging capabilities.
• Provides a rapid and effective solution for whole-cell volumetric nanoscale imaging.
• Opens new avenues for studying complex cellular structures in their native 3D context.