Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Area of Science:

• Biophysics
• Optical Microscopy
• Cellular Biology

Background:

• Achieving high 3D spatial resolution is crucial for understanding cellular organization.
• 4Pi nanoscopy methods use opposing objectives and fluorescence state switching to improve resolution but are limited by optical aberrations in thick samples.

Purpose of the Study:

• To develop an advanced adaptive optics strategy for iso-stimulated emission depletion (iSTED) nanoscopy.
• To overcome the limitations of optical aberrations in thick specimens for super-resolution imaging.

Main Methods:

• Implementation of an advanced adaptive optics system within an iSTED nanoscope.
• Utilizing a beam path geometry with two opposing objectives for optimal resolution.
• Application of adaptive optics to correct for optical aberrations deep within aberrating specimens.

Main Results:

• Achieved sub-50-nm isotropic resolution in 3D.
• Successfully imaged previously inaccessible structures like neuronal synapses and ring canals in tissue.
• Demonstrated the general applicability of the adaptive optics scheme to similar microscopy setups.

Conclusions:

• The developed adaptive optics strategy significantly enhances iSTED nanoscopy capabilities.
• This method enables high-resolution 3D imaging in thick, aberrating biological specimens.
• The adaptive optics approach is broadly applicable to dual-objective microscopes for deep-tissue imaging.