Deep Volumetric Super-Resolution Imaging in Thick Biological Specimens With Sparse Scanning SIM

Area of Science:

• Biophotonics and advanced microscopy techniques.
• Development of novel imaging modalities for biological research.

Background:

• High-resolution fluorescence microscopy faces challenges in deep, thick specimens due to light scattering and limited penetration.
• Structured illumination microscopy (SIM) offers super-resolution but is sensitive to scattering, hindering volumetric imaging.
• Existing methods struggle with deep imaging in complex biological environments.

Purpose of the Study:

• To develop a super-resolution microscopy technique for deep imaging in thick, live specimens.
• To overcome the limitations of conventional SIM in scattering-prone biological samples.
• To enable high-contrast, volumetric visualization of dense biological structures.

Main Methods:

• Introduction of sparse scanning SIM (SS-SIM), integrating rapid laser scanning with pixel-addressed intensity modulation and sCMOS detection.
• Utilization of either one- or two-photon excitation for versatile deep imaging applications.
• Implementation with water-immersion objectives to achieve imaging depths up to 300/600 µm.

Main Results:

• SS-SIM achieved a spatial resolution of approximately 150 nm.
• Maintained a 1.6/1.7-fold (lateral/axial) resolution enhancement over wide-field microscopy within the imaging depth.
• Successfully enabled volumetric visualization of dense biological samples, including thick tissues, organoids, and small organisms.

Conclusions:

• SS-SIM provides a robust and simple method for deep super-resolution imaging.
• This technique significantly expands the applicability of SIM to challenging biological samples.
• SS-SIM offers a practical solution for high-contrast volumetric imaging in complex biological environments.