Supercritical angle fluorescence for enhanced axial sectioning in STED microscopy

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Supercritical angle fluorescence (SAF) imaging detects fluorophores near interfaces by capturing evanescent near-field emission that becomes propagative beyond the critical angle.
• This technique allows for high-resolution imaging of cellular membranes based on fluorophore proximity to the coverslip.
• Conventional microscopy often struggles with precise axial localization of structures within biological samples.

Purpose of the Study:

• To demonstrate subwavelength axial sectioning on biological samples.
• To combine stimulated emission depletion (STED) microscopy with supercritical angle fluorescence (SAF) detection for enhanced axial resolution.
• To investigate the feasibility of implementing STED-SAF for improved imaging of cellular structures.

Main Methods:

• Utilized a stimulated emission depletion (STED) microscope.
• Integrated supercritical angle fluorescence (SAF) detection capabilities.
• Employed filtering of SAF emission from undercritical angle fluorescence (UAF) emission in the objective lens's back focal plane.

Main Results:

• Achieved subwavelength axial sectioning on biological samples.
• Demonstrated a significant gain in axial resolution by combining STED and SAF.
• Showcased that STED-SAF implementation requires only a modification in the detection path, not the STED beam path.

Conclusions:

• STED-SAF microscopy enables nanometer-scale axial sectioning of fluorescent emitters near the coverslip.
• The combination of STED and SAF offers a straightforward enhancement in axial resolution for microscopy.
• STED-SAF is a widely implementable technique with minimal alterations to existing STED microscope setups.