Low-temperature fabrication and characterization of a symmetric hybrid organic–inorganic slab waveguide for evanescent light microscopy

Abstract

Organic and inorganic solid materials form the building blocks for most of today’s high-technological instruments and devices. However, challenges related to dissimilar material properties have hampered the synthesis of thin-film devices comprised of both organic and inorganic films. We here give a detailed description of a carefully optimized processing protocol used for the construction of a three-layered hybrid organic–inorganic waveguide-chip intended for combined scattering and fluorescence evanescent-wave microscopy in aqueous environments using conventional upright microscopes. An inorganic core layer (SiO2 or Si3N4), embedded symmetrically in an organic cladding layer (CYTOP), aids simple, yet efficient in-coupling of light, and since the organic cladding layer is refractive index matched to water, low stray-light (background) scattering of the propagating light is ensured. Another major advantage is that the inorganic core layer makes the chip compatible with multiple well-established surface functionalization schemes that allows for a broad range of applications, including detection of single lipid vesicles, metallic nanoparticles or cells in complex environments, either label-free—by direct detection of scattered light—or by use of fluorescence excitation and emission. Herein, focus is put on a detailed description of the fabrication of the waveguide-chip, together with a fundamental characterization of its optical properties and performance, particularly in comparison with conventional epi illumination. Quantitative analysis of images obtained from both fluorescence and scattering intensities from surface-immobilized polystyrene nanoparticles in suspensions of different concentrations, revealed enhanced signal-to-noise and signal-to-background ratios for the waveguide illumination compared to the epi-illumination.

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein

“…Waveguide Chip Fabrication and Surface Modification. The fabrication of the waveguide device is described in detail elsewhere (67). Briefly, a 400-nm-thick silica core layer (spin-on-glass, IC1-200 from Futurrex, Inc.), embedded in a 7μm-thick cladding layer of fluorinated polymer (CYTOP, CTX-809AP2 from AGC Chemicals, ASAHI Glass Co., Ltd.), was fabricated on a standard Si(100) 4-inch wafer.…”

Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins

“…Agnarsson et al introduced a waveguide chip that was capable of single particle fluorescence and scattering measurements (Figure A). , In this technique, the sample was exposed to the core of the waveguide by etching a sample well into the polymer cladding layer on top of the silica core (Figure A). Light was coupled in to the single-mode waveguide via a single-mode fiber, which generated an evanescent field that penetrated the core–water interface.…”

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles

“…The waveguide platform allows for substantial reduction of the background signal by matching the refractive index of the waveguide cladding to the protein-containing buffer solution, providing label-free observation of the scattering signal from individual surface-bound NPs. 14 To demonstrate the application of this technique in the context under consideration, we measured specific binding of streptavidin (SA) and antibiotin-IgG antibodies (antibiotin) to surface-bound biotin-modified lipid vesicles. To verify that the observed changes in the scattering signal originate from protein binding, both proteins were fluorescently labeled, which in addition enabled a direct comparison between the signal-tonoise ratios for the two modes of operation.…”

“…The waveguide platform allows for substantial reduction of the background signal by matching the refractive index of the waveguide cladding to the protein-containing buffer solution, providing label-free observation of the scattering signal from individual surface-bound NPs. 14 …”

“…Here, we take a step toward addressing the challenge of quantitative characterization of protein binding kinetics to surface-bound NPs using evanescent waveguide microscopy, enabling real-time signal acquisition from simultaneously recorded fluorescence and label-free light-scattering signals. The waveguide platform allows for substantial reduction of the background signal by matching the refractive index of the waveguide cladding to the protein-containing buffer solution, providing label-free observation of the scattering signal from individual surface-bound NPs …”