学术文献库

Total internal reflection fluorescence microscopy (TIRF) has enabled low-background, live-cell friendly imaging of cell surfaces and other thin samples thanks to the shallow penetration of the evanescent light field into the sample. The implementation of TIRF on optical waveguide chips (c-TIRF) has overcome historical limitations on the magnification and field of view (FOV) compared to lens-based TIRF, and further allows the light to be guided in complicated patterns that can be used for advanced imaging techniques or selective stimulation of the sample. However, the opacity of the chips themselves has thus far precluded their use on inverted microscopes and complicated sample preparation and handling. In this work, we introduce a new platform for c-TIRF imaging based on a transparent substrate, which is fully compatible with sample handling and imaging procedures commonly used with a standard #1.5 glass coverslip, and is fabricated using standard complementary metal-oxide-semiconductor (CMOS) techniques, which can easily be scaled up for mass production. We demonstrate its performance on synthetic and biological samples using both upright and inverted microscopes, and show how it can be extended to super-resolution applications, achieving a resolution of 116 nm using super resolution radial fluctuations (SRRF). These new chips retain the scalable FOV of opaque chip-based TIRF and the high axial resolution of TIRF, and have the versatility to be used with many different objective lenses, microscopy methods, and handling techniques. We thus see c-TIRF as a technology primed for widespread adoption, increasing both TIRF's accessibility to users and the range of applications that can benefit from it.