Abstract
Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that, in comparison to confocal microscopy, does not require a trade-off between resolution, speed, and photodamage. Here, we introduce a waveguide platform for chip-based 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. The platform is fabricated using standard complementary metal-oxide-semiconductor 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. These transparent chips retain the scalable field of view 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 see this as a technology primed for widespread adoption, increasing both TIRF’s accessibility to users and the range of applications that can benefit from it.
Highlights
Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that, in comparison to confocal microscopy, does not require a trade-off between resolution, speed, and photodamage
While objective lens-based TIRF is restricted to using a high NA lens, limiting its field of view (FOV), the evanescent field generated by waveguides is independent of the imaging pathway, enabling them to be used with any imaging objective on a standard microscope[13,17,19]
The inherent variation in the illumination pattern in broad waveguides makes the c-TIRF platform a natural partner for fluctuation-based super-resolution techniques, and the high RI of the waveguide enables even smaller patterns to be generated for fluctuations than is possible with free-space optics
Summary
Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that, in comparison to confocal microscopy, does not require a trade-off between resolution, speed, and photodamage. 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 These transparent chips retain the scalable field of view 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 see this as a technology primed for widespread adoption, increasing both TIRF’s accessibility to users and the range of applications that can benefit from it. Most chip-based microscopy has been performed on waveguides fabricated on top of opaque substrates[15,16,17,19,20]
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