Large Field-of-View Super-Resolution Optical Microscopy Based on Planar Polymer Waveguides

Abstract

Planar photonic waveguides enable ultrathin sample illumination in fluorescence microscopy over exceedingly large fields of view. Their fabrication has been based on hard coatings requiring sputter deposition and ion-beam lithography, making volume production cumbersome and thereby limiting their availability. Additionally, they are typically fabricated on top of opaque silicon wafers, which restricts the use to upright microscopes. Here, we present a low-cost photonic waveguide chip based on standard 170 μm thick glass coverslips coated with a micrometer-thin layer of EpoCore, a photoresist polymer with a high refractive index. These chips enable wide-field excitation for fluorescence microscopy and nanoscopy on inverted microscopes using fluorescence detection through the transparent substrate. Channel waveguides with varying widths provide uniform near-field illumination by evanescent waves for fields of view up to the millimeter scale. We demonstrate multicolor fluorescence excitation resulting in high-contrast immunofluorescence and super-resolution imaging with a resolution of approximately 60 nm.

Dual-color single molecule localization microscopy on transparent polymer waveguide chips

“…The localization intensity also decreased from a starting value of 700 photons and 500 photons for 568 nm and 647 nm excitation, respectively, to ∼400 photons for both wavelengths. As we saw for bulk autofluorescence in [15], we expect the autofluorescent impurities to slowly recover over the course of multiple days. This shows that autofluorescence in the polymer is readily suppressed after the first few minutes of any SMLM experiment and will not lead to artefacts in the reconstructed images.…”

“…We produce EpoCore waveguide chips with 1.2 µm core height as reported earlier [15]. The chips are first visually inspected by coupling the beam into a set of waveguides, then cleaned by submersion in a ultrasonicator bath containing 0.05% sodium dodecyl sulfate (SDS) for 5 minutes.…”

“…In addition to a further increase in production costs, the brittle nature of this material makes handling of such chips extremely delicate, again limiting their widespread use. Recently, we introduced an affordable and highly efficient transparent photonic waveguide chip based on the negative photoresist polymer EpoCore which is spin-coated on top of ordinary #1.5 coverslips [15,16]. Transparent polymer waveguide chips used in combination with an inverted microscopes enabled waveguide-TIRF imaging of biological samples with an unprecedented large field-of-view (FOV).…”

Label-free incoherent super-resolution optical microscopy

“…This PL of Si3N4 waveguide does not exhibit any bleaching effect over long periods of times (Fig. 2f), as opposed to the autofluorescence in polymer waveguides [51] and varies linearly with the excitation power (Fig. 2g).…”

Transmission structured illumination microscopy with tunable frequency illumination using tilt mirror assembly

“…This limitation appears because of the inherent trade-off between the resolution and the FOV of the standard imaging system. Because of this dependence, the next level superresolution techniques have been dealing with large FOV imaging while improving the resolution 33,34 . Recently, a theoretical approach 35 was reported to overcome this limitation by decoupling the illumination path from the collection path using a transmission type tilt-mirror configuration.…”