Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

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Super-resolution microscopy demystified

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells. Nanoscopy on a chip makes it possible to perform super-resolution imaging of biological specimens with a wide field-of-view.

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Chip-based wide field-of-view nanoscopy

Offering open-access silicon photonics-based technologies has played a pivotal role in unleashing this technology from research laboratories to industry. Fabless enterprises rely on the open-access of these technologies for their product development. In the last decade, a diverse set of open-access technologies with medium and high technology readiness levels have emerged. This paper provides a review of the open-access silicon and silicon nitride photonic IC technologies offered by the pilot lines of European research institutes and companies. The paper also highlights upcoming features of these platforms and discusses how they address the long-term market needs.

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Open-Access Silicon Photonics Platforms in Europe

Propulsive effects of light, which often remain unnoticed in our daily-life experience, manifest themselves on spatial scales ranging from subatomic to astronomical. Light-mediated forces can indeed confine individual atoms, cooling their effective temperature very close to absolute zero, as well as contribute to cosmological phenomena such as the formation of stellar planetary systems. In this review, we focus on the transport processes that light can initiate on small spatial scales. In particular, we discuss in depth various light-induced mechanisms for the controlled transport of microscopic particles; these mechanisms rely on the direct transfer of momentum between the particles and the incident light waves, on the combination of optical forces with external forces of other nature, and on light-triggered phoretic motion. After a concise theoretical overview of the physical origins of optical forces, we describe how these forces can be harnessed to guide particles either in continuous bulk media or in the proximity of a constraining interface under various configurations of the illuminating light beams (radiative, evanescent, or plasmonic fields). Subsequently, we introduce particle transport techniques that complement optical forces with counteracting forces of non-optical nature. We finally discuss particle actuation schemes where light acts as a fine knob to trigger and/or modulate phoretic motion in spatial gradients of non-optical (e.g., electric, chemical, or temperature) fields. We conclude by outlining possible future fundamental and applied directions for research in light-induced particle transport. We believe that this comprehensive review can inspire diverse, interdisciplinary scientific communities to devise novel, unorthodox ways of assembling and manipulating materials with light.

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Perspective on light-induced transport of particles: from optical forces to phoretic motion

Structured illumination microscopy (SIM) enables live-cell super-resolution imaging of subcellular structures at high speeds. At present, linear SIM uses free-space optics to illuminate the sample with the desired light patterns; however, such arrangements are prone to misalignment and add cost and complexity to the microscope. Here, we present an alternative photonic chip-based two-dimensional SIM approach (cSIM) in which the conventional glass sample slide in a microscope is replaced by a planar photonic chip that importantly both holds and illuminates the specimen. The photonic chip reduces the footprint of the light illumination path of SIM to around 4 × 4 cm2. An array of optical waveguides on the chip creates standing wave interference patterns at different angles, which illuminate the sample via evanescent fields. High-refractive-index silicon nitride waveguides allow a 2.3 times enhancement in imaging spatial resolution, exceeding the usual 2 times limit of SIM. In summary, cSIM offers a simple, stable and affordable approach for performing two-dimensional super-resolution imaging over a large field of view. The use of a photonic integrated circuit to both hold a biological sample and generate the necessary light patterns for structured illumination microscopy promises convenient super-resolution imaging.

Structured illumination microscopy using a photonic chip

Structured illumination microscopy (SIM) enables live cell, super-resolution imaging at high speeds. SIM uses sophisticated optical systems to generate pre-determined excitation light patterns, and reconstruction algorithms to enhance the resolution by up to a factor of two. The optical set-up of SIM relies on delicate free-space optics to generate the light patterns, and a high numerical aperture objective lens to project the pattern on the sample. These arrangements are prone to miss-alignment, often with high costs, and with the final resolution-enhancement being limited by the numerical aperture of the collection optics. Here, we present a photonic-chip based total internal reflection fluorescence (TIRF)-SIM that greatly reduces the complexity of the optical setup needed to acquire TIRF-SIM images. This is achieved by taking out the light delivery from the microscope and transferring it to a photonic-chip. The conventional glass slide is replaced by the planar photonic chip, that both holds and illuminates the specimen. The chip is used to create a standing wave interference pattern, which illuminates the sample via evanescent fields. The phase of the interference pattern is controlled by the use of thermo-optical modulation, leaving the footprint of the light illumination path for the SIM system to around 4 by 4 cm$^2$. Furthermore, we show that by the use of the photonic-chip technology, the resolution enhancement of SIM can be increased above that of the conventional approach. In addition, by the separation of excitation and collection light paths the technology opens the possibility to use low numerical objective lenses, without sacrificing on the SIM resolution. Chip-based SIM represents a simple, stable and affordable approach, which could enable widespread penetration of the technique and might also open avenues for high throughput optical super-resolution microscopy.

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 (Si3N4) 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. Si3N4 material is finally shown to be biocompatible for growing and imaging living cells.

Silicon nitride waveguide platform for fluorescence microscopy of 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 × 0.5 mm, using a low magnification and high N.A. objective lens. 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.

Øystein Ivar Helle

Papers

Chip-based wide field-of-view nanoscopy

Structured illumination microscopy using a photonic chip

Structured illumination microscopy using a photonic chip

Silicon nitride waveguide platform for fluorescence microscopy of living cells.

Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale