Improved microscopy with ultraviolet surface excitation (MUSE) using high-index immersion illumination

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Pathology Imaging

Background:

• Microscopy with ultraviolet surface excitation (MUSE) typically yields thicker optical sections than physical sectioning.
• This leads to increased background fluorescence and higher feature density in MUSE imaging.
• Existing MUSE methods are limited in optical sectioning capability for detailed tissue analysis.

Purpose of the Study:

• To reduce the optical sectioning thickness of MUSE.
• To improve resolution and contrast in imaging surgical specimens.
• To develop a MUSE system with a wider field of view and robustness to surface irregularities.

Main Methods:

• Implemented high-index immersion with angled illumination to reduce optical sectioning thickness.
• Designed a novel objective dipping cap and waveguide-based MUSE illuminator.
• Quantified optical sectioning thickness reduction and field of view using the new system.
• Applied deconvolution and focal stacking for further image enhancement.

Main Results:

• Achieved an e^-1 section thickness reduction to 6.67 µm in tissue.
• Demonstrated a 6 mm² field of view, exceeding conventional pathology objectives.
• Showcased improved resolution and contrast for imaging irregular surgical specimen surfaces.
• Validated the effectiveness of high-index immersion and waveguide illumination in MUSE.

Conclusions:

• High-index immersion with angled illumination effectively reduces MUSE optical sectioning thickness.
• The novel waveguide-based illuminator enables wider field of view and improved imaging quality.
• This enhanced MUSE technique offers robust imaging for surgical pathology, improving diagnostic potential.