High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using...

Area of Science:

• Optical microscopy
• Super-resolution imaging
• Nanotechnology

Background:

• Total internal reflection fluorescence (TIRF) microscopy enhances contrast in super-resolution imaging via optical sectioning.
• Conventional TIRF microscopy is limited by small fields of view and low throughput due to high numerical aperture objectives for excitation and collection.

Purpose of the Study:

• To introduce and demonstrate chip-based nanoscopy, a novel method for generating TIRF excitation using optical waveguides.
• To showcase the advantages of decoupled excitation and collection pathways for enhanced imaging capabilities.

Main Methods:

• Implementation of chip-based nanoscopy in an existing microscope setup.
• Utilizing optical waveguides for TIRF excitation.
• Imaging liver sinusoidal endothelial cells (LSECs) using direct stochastic optical reconstruction microscopy (dSTORM).

Main Results:

• Achieved large field of view TIRF images using a low magnification lens, decoupling excitation and collection.
• Demonstrated high-throughput imaging of a 500 µm x 500 µm area with 76 nm resolution.
• Obtained resolution comparable to traditional super-resolution microscopes.

Conclusions:

• Chip-based nanoscopy provides a compact, retrofittable solution for high-throughput 2D super-resolution imaging.
• The technique allows for imaging with a large field of view and potential for integration with other on-chip optical techniques for multi-modal analysis.