Maximising imaging volumes of expanded tissues for inverted fluorescence microscopy

Area of Science:

• Biomedical Imaging
• Microscopy Techniques
• Cell Biology

Background:

• Expansion microscopy (ExM) enhances nanoscale imaging by physically enlarging biological samples within a hydrogel.
• Conventional inverted confocal microscopy faces challenges with deep imaging of ExM samples due to limited objective working distances in aqueous environments.

Purpose of the Study:

• To develop a practical and cost-effective method for high-resolution, deep volumetric imaging of expansion microscopy samples using standard inverted microscopes.
• To address the challenges posed by the refractive index mismatch and limited working distance in imaging enlarged biological samples.

Main Methods:

• Utilized an inverted water-dipping objective combined with a refractive-index-matched optical path created by fluorinated ethylene propylene (FEP) film.
• Employed a stable immersion setup using FEP film, Immersol W, water, and an FEP-based imaging dish.
• Characterized the optical path using point spread function (PSF) measurements and simulations.

Main Results:

• The FEP film introduced correctable defocus-like wavefront aberrations, enabling deep imaging.
• Achieved sub-micron lateral and axial resolution with stable image quality up to depths exceeding 800 µm.
• Demonstrated successful imaging of 4×-expanded U2OS cells and human cerebral organoids.

Conclusions:

• The developed system offers a low-cost, plug-and-play solution for high-resolution volumetric imaging of ExM samples.
• This approach enhances the accessibility of deep tissue imaging for expanded biological specimens using standard inverted microscopes.
• The method effectively overcomes previous limitations in imaging depth and resolution for expanded samples.