Deep-hole array structure for highly sensitive fluorescence imaging of cells via inverted microscopy

Area of Science:

• Nanophotonics
• Biomedical Engineering
• Microscopy

Background:

• Inverted fluorescence microscopy is ideal for studying cell membranes at interfaces.
• Surface-plasmon-mediated enhancement often reduces light transmission, hindering microscopy.
• Homogeneous fluorescence enhancement is crucial for high-sensitivity, wide-field imaging.

Purpose of the Study:

• To develop a plasmonic chip for enhanced fluorescence detection with inverted microscopy.
• To overcome limitations of reduced light transmission in conventional nanostructure-based enhancement.
• To enable sensitive imaging of membrane proteins and other targets at the cell-substrate interface.

Main Methods:

• Fabrication of a periodic deep-hole array (DHA) plasmonic chip with 422 nm depth.
• Utilizing the enlarged inner areas and sidewalls of deep holes to increase optical transmittance.
• Comparing fluorescence intensity and imaging capabilities with conventional hole-array structures and cover slips.

Main Results:

• The DHA structure achieved over 50-fold higher optical transmittance than conventional structures.
• A ~70-fold increase in fluorescence intensity was observed for 0.2 µm fluorescent particles.
• Successfully imaged EpCAMs on breast cancer cell membranes, which were previously undetectable.

Conclusions:

• The DHA plasmonic chip provides a powerful platform for highly sensitive fluorescence detection.
• This technology enhances inverted fluorescence microscopy for imaging cellular components.
• Potential applications include single-nanoparticle analysis and advanced cellular bioimaging.