Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Confocal spinning-disk (CSD) microscopy is a widely used technique for live-cell imaging.
• Achieving super-resolution often requires specialized and expensive equipment.
• There is a need for accessible super-resolution methods compatible with existing microscopes.

Purpose of the Study:

• To demonstrate the conversion of a conventional confocal spinning-disk microscope into a super-resolution image scanning microscopy (ISM) system.
• To achieve nearly doubled resolution without altering the microscope's optical or mechanical components.
• To showcase the applicability of this adapted ISM technique for detailed biological imaging.

Main Methods:

• Utilized the intrinsic properties of a CSD microscope for stroboscopic illumination.
• Generated an array of excitation foci moved across the sample by phase modulation.
• Applied image scanning microscopy (ISM) principles to reconstruct a high-resolution image.

Main Results:

• Achieved nearly doubled resolution compared to conventional CSD microscopy.
• Successfully imaged single nuclear pore complexes and GFP-Tau protein aggregates in 3D.
• Demonstrated multicolor ISM imaging of cytoskeletal proteins and multi-stained 3D samples (MitoTracker, Hoechst).

Conclusions:

• A conventional CSD microscope can be readily adapted for super-resolution imaging using ISM.
• This method provides a cost-effective approach to enhance imaging resolution for cell biology.
• The adapted ISM technique is versatile and applicable to various biological samples and research questions.