Optical microscopy based on waveguide chips significantly reduces the complexity of the entire optical setup, enabling miniaturization by completely removing the excitation light path from the microscope. Instead, waveguides which tightly confine the guided light by total internal reflection due to a high refractive index contrast (HIC) to the surrounding media such as water and cells are used to deliver the illumination light to the sample. The evanescent field on top of the waveguide can be utilized for total internal reflection fluorescence (TIRF) excitation over an almost arbitrarily wide FOV that is intrinsically independent of the detection objective lens and in principle only limited by the waveguide design. Evanescent field excitation using waveguides was first introduced by Grandin et al. 17 , where a slab waveguide was used to generate an evanescent field over the large stretch of the waveguide chip. Slab waveguides (Fig. 1a) Here, we demonstrate waveguide chip-based super-resolution fluorescence imaging by two complementary approaches using ESI and dSTORM. The high intensity in the evanescent field generated by the HIC waveguide material is used for optical switching of fluorophores as required by dSTORM. In addition, the intrinsically multi-mode interference pattern within the waveguide is used to generate fluctuating intensity patterns for ESI. To demonstrate the applicability of waveguide chip-based super-resolution microscopy we visualize the connection of the actin cytoskeleton and plasma membrane fenestrations in liver sinusoidal endothelial cells (LSECs). RESULTS Chip-based single molecule localization microscopyThe performance of chip-based dSTORM is shown by imaging immunostained microtubules in rat LSECs 26 plated directly on the waveguide (Fig. 2a). Measuring the lateral profile along one straight microtubule filament reveals a hollow structure 27 which has been used earlier in localization microscopy as a benchmark sample [28][29][30] , discussed in detail in 31 . This shows a resolution of better than 50 nm ( Fig. 2b), confirmed by full-width-at-half-maximum (FWHM) values, localization precision 32 , and Fourier ring correlation 33,34 (FRC) calculations ( Supplementary Fig. 1). The resolution capability was further investigated by using DNA origami nanorulers that provide markers at (50 ± 5) nm distance as references. These can be clearly resolved in chip-based dSTORM (Fig. 2c,d, Supplementary Fig. 2) which shows a comparable performance to the widely used architecture of an inverted TIRF dSTORM setup (Fig. 2c,d, Supplementary Fig. 3).As an advantage over conventional setups, waveguide chip-based nanoscopy greatly benefits from the fact that the fluorescence excitation is independent of the detection objective lens. As fluorescence is excited by the evanescent field of the waveguide, the technique provides optical sectioning and excellent signal to background ratios at penetration depths below 200 nm ( Supplementary Fig. 4, Supplementary Fig. 5, Video 1), similar to objective-based TIRF...
Waveguide chip-based microscopy reduces the complexity of total internal reflection fluorescence (TIRF) microscopy, and adds features like large field of view illumination, decoupling of illumination and collection path and easy multimodal imaging. However, for the technique to become widespread there is a need of low-loss and affordable waveguides made of high-refractive index material. Here, we develop and report a low-loss silicon nitride (Si 3 N 4 ) waveguide platform for multi-color TIRF microscopy. Single mode conditions at visible wavelengths (488-660 nm) were achieved using shallow rib geometry. To generate uniform excitation over appropriate dimensions waveguide bends were used to filter-out higher modes followed by adiabatic tapering. Si 3 N 4 material is finally shown to be biocompatible for growing and imaging living cells.
Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm x 0.5 mm, using a low magnification and high N.A. objective l ens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.
Optical waveguides can be used to trap and transport microparticles. The particles are held close to the waveguide surface by the evanescent field and propelled forward. We propose a new technique to lift and trap particles above the surface of the waveguides. This is made possible by a gap between two opposing, planar waveguides. The field emitted from each of the waveguide ends diverge fast, away from the substrate and into the cover-medium. By combining two fields propagating at an angle upwards and coming from opposite sides of a gap, particles can be stably lifted and trapped at the crossing of the two fields. Thus, particles are transported by waveguides leading to a gap, where they are lifted away from the substrate and trapped. The experiments are supported by numerical simulations of the forces on the micro-particles. Fluorescence imaging is used to track the particles in 3D with a precision of 50 nm.
Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.
Photonic-chip based TIRF illumination has been used to demonstrate several on-chip optical nanoscopy methods. The sample is illuminated by the evanescent field generated by the electromagnetic wave modes guided inside the optical waveguide. In addition to the photokinetics of the fluorophores, the waveguide modes can be further exploited for introducing controlled intensity fluctuations for exploitation by techniques such as super-resolution optical fluctuation imaging (SOFI). However, the problem of nonuniform illumination pattern generated by the modes contribute to artifacts in the reconstructed image. To alleviate this problem, we propose to perform Haar wavelet kernel (HAWK) analysis on the original image stack prior to the application of (SOFI). HAWK produces a computational image stack with higher spatio-temporal sparsity than the original stack. In the case of multimoded non-uniform illumination patterns, HAWK processing breaks the mode pattern while introducing spatio-temporal sparsity, thereby differentially affecting the non-uniformity of the illumination. Consequently, this assists nanoscopy methods such as SOFI to better support super-resolution, which is otherwise compromised due to spatial correlation of the mode patterns in the raw image. Furthermore, applying HAWK prior to SOFI alleviates the problem of artifacts due to non-uniform illumination without degrading temporal resolution. Our experimental results demonstrate resolution enhancement as well as reduction in artifacts through the combination of HAWK and SOFI.
Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 µm x 500 µm region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multimodal analysis.
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