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...

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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.

Waveguide trapping has emerged as a useful technique for parallel and planar transport of particles and biological cells and can be integrated with lab-on-a-chip applications. However, particles trapped on waveguides are continuously propelled forward along the surface of the waveguide. This limits the practical usability of the waveguide trapping technique with other functions (e.g. analysis, imaging) that require particles to be stationary during diagnosis. In this paper, an optical waveguide loop with an intentional gap at the centre is proposed to hold propelled particles and cells. The waveguide acts as a conveyor belt to transport and deliver the particles/cells towards the gap. At the gap, the diverging light fields hold the particles at a fixed position. The proposed waveguide design is numerically studied and experimentally implemented. The optical forces on the particle at the gap are calculated using the finite element method. Experimentally, the method is used to transport and trap micro-particles and red blood cells at the gap with varying separations. The waveguides are only 180 nm thick and thus could be integrated with other functions on the chip, e.g. microfluidics or optical detection, to make an on-chip system for single cell analysis and to study the interaction between cells.

Semen quality assessed by sperm count and sperm cell characteristics such as morphology and motility, is considered to be the main determinant of men’s reproductive health. Therefore, sperm cell selection is vital in assisted reproductive technology (ART) used for the treatment of infertility. Conventional bright field optical microscopy is widely utilized for the imaging and selection of sperm cells based on the qualitative analysis by experienced clinicians. In this study, we report the development of a highly sensitive quantitative phase microscopy (QPM) using partially spatially coherent light source, which is a label-free, non-invasive and high-resolution technique to quantify various biophysical parameters. The partial spatial coherence nature of light source provides a significant improvement in spatial phase sensitivity and hence reconstruction of the phase of the entire sperm cell is demonstrated, which was otherwise not possible using highly spatially coherent light source. High sensitivity of the system enables quantitative phase imaging of the specimens having very low refractive index contrast with respect to the medium like tail of the sperm cells. Further, it also benefits with accurate quantification of 3D-morphological parameters of sperm cells which might be helpful in the infertility treatment. The quantitative analysis of more than 2500 sperm cells under hydrogen peroxide (H 2 O 2 ) induced oxidative stress condition is demonstrated. It is further correlated with motility of sperm cell to study the effect of oxidative stress on healthy sperm cells. The results exhibit a decrease in the maximum phase values of the sperm head as well as decrease in the sperm cell’s motility with increasing oxidative stress, i.e., H 2 O 2 concentration. Various morphological and texture parameters were extracted from the phase maps and subsequently support vector machine (SVM) based machine learning algorithm is employed for the classification of the control and the stressed sperms cells. The algorithm achieves an area under the receiver operator characteristic (ROC) curve of 89.93% based on the all morphological and texture parameters with a sensitivity of 91.18%. The proposed approach can be implemented for live sperm cells selection in ART procedure for the treatment of infertility.

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.

Our ability to generate new distributions of light has been remarkably enhanced in recent years. At the most fundamental level, these light patterns are obtained by ingeniously combining different electromagnetic modes. Interestingly, the modal superposition occurs in the spatial, temporal as well as spatio-temporal domain. This generalized concept of structured light is being applied across the entire spectrum of optics: generating classical and quantum states of light, harnessing linear and nonlinear light-matter interactions, and advancing applications in microscopy, spectroscopy, holography, communication, and synchronization. This Roadmap highlights the common roots of these different techniques and thus establishes links between research areas that complement each other seamlessly. We provide an overview of all these areas, their backgrounds, current research, and future developments. We highlight the power of multimodal light manipulation and want to inspire new eclectic approaches in this vibrant research community.

Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA - their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.