Abstract
Super-resolution microscopies based on the localization of single molecules have been widely adopted due to their demonstrated performance and their accessibility resulting from open software and simple hardware. The PAINT method for localization microscopy offers improved resolution over photoswitching methods, since it is less prone to sparse sampling of structures and provides higher localization precision. Here, we show that waveguides enable increased throughput and data quality for PAINT, by generating a highly uniform ~100 × 2000 µm2 area evanescent field for TIRF illumination. To achieve this, we designed and fabricated waveguides optimized for efficient light coupling and propagation, incorporating a carefully engineered input facet and taper. We also developed a stable, low-cost microscope and 3D-printable waveguide chip holder for easy alignment and imaging. We demonstrate the capabilities of our open platform by using DNA-PAINT to image multiple whole cells or hundreds of origami structures in a single field of view.
Highlights
Super-resolution microscopies based on the localization of single molecules have been widely adopted due to their demonstrated performance and their accessibility resulting from open software and simple hardware
Waveguide total internal reflection fluorescence (TIRF) excitation for fluorescence microscopy has previously been demonstrated with high-index waveguide cores fabricated from either tantalum pentoxide (Ta2O5)[35,36] or silicon nitride (Si3N4)[33,37], including being used to perform direct STORM imaging
Increasing the throughput of localization microscopy has previously been achieved by automating acquisitions to image multiple FOV51 or multiple wells under different conditions[52]
Summary
Super-resolution microscopies based on the localization of single molecules have been widely adopted due to their demonstrated performance and their accessibility resulting from open software and simple hardware. A too-low density of localizations results in an undersampled structure, insufficient to resolve its organization even in the case of nanometric localization precisions[9] This practical limitation of stochastic photoswitching is circumvented by methods that instead use binding and dissociation of fluorescent probes, such as ‘points accumulation in nanoscale topography’ (PAINT)[10] and extensions thereof which include complementation between target and imager DNA strands in DNA-PAINT11,12 and protein-fragment probes in ‘integrating exchangeable single-molecule localization’ (IRIS)[13]. It requires axial optical sectioning to reject the background signal from fluorophores in solution This can be mitigated in the case of fluorescence enhancement upon binding as for fluorogenic dyes[10], quenching of unbound probes[17] or Förster resonance energy transfer-(FRET) PAINT18,19, but at the cost of reduced labeling flexibility, increased sample preparation complexity and a potential reduction in localization precision[20,21]. PAINT generally requires an integration time per localization more than 10x longer than for stochastic photoswitching[28]
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