![]() In addition to achieving a sub-diffraction localization precision of 75 nm, chip-based SMLM was able to do this with an unprecedented FOV of 0.5 × 0.5 mm 2, approximately 100 times larger than objective lens-based techniques 17, 18. The increased relative intensity due to confinement of the light within a thin waveguide is particularly well-suited for these power-hungry techniques. Furthermore, waveguide chips have been extended to super-resolution modalities, including single-molecule localization microscopy (SMLM) 17, 20, entropy-based super-resolution imaging (ESI) 18, and structured illumination microscopy (SIM) 20. While objective lens-based TIRF is restricted to using a high NA lens, thus 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. These photonic chips are fabricated using technology similar to computer chips, and thus have the potential to be mass-produced at low cost. The angle necessary for TIRF has traditionally been achieved using a high numerical aperture (NA) objective lens 1, 10, 11, through a prism 1, 4, 11, 12, through the use of grating couplers 13, 14 ( ) and more recently by coupling into the side facet of optical waveguide chips 15, 16, 17, 18, 19, 20, 21. When light is directed into an interface between media with a high index of refraction contrast (HIC) at a sufficiently high angle, the light is totally reflected within that interface while the light itself does not escape from the high index material, an evanescent field is generated along the surface that the light travels 2, 9. The enabling mechanism of TIRF is the generation of a thin, exponentially decaying layer of light at a surface, called the evanescent field. ![]() TIRF’s ability to focus exclusively on a thin layer at the surface of the cell has made it an excellent tool for studying, among others, the dynamics of focal adhesions 5, the inner workings of endocytosis 6, the kinetics of cell surface receptors 7, and docking of synaptic vesicles with neurons 8. Additionally, because TIRF is a widefield technique, acquisition of a full image can occur within milliseconds. In TIRF, only the bottom ~100 nm of the sample is excited 1, 2, 3, 4, 5, which improves the axial resolution, eliminates out-of-focus light, and protects the bulk of the sample from photodamage. Total internal reflection fluorescence (TIRF) microscopy, however, is a rare example of a method, which improves all three simultaneously, compared to confocal microscopy. 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.Ī common trade-off in microscopy is between resolution, speed, and photodamage or photobleaching. 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 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. The platform is fabricated using standard complementary metal-oxide-semiconductor techniques which can easily be scaled up for mass production. 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 timestamp is only as accurate as the clock in the camera, and it may be completely wrong.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. If the file has been modified from its original state, some details such as the timestamp may not fully reflect those of the original file. This file contains additional information such as Exif metadata which may have been added by the digital camera, scanner, or software program used to create or digitize it.
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