Identification of extracellular vesicles from their Raman spectra via self-supervised learning.

Extracellular vesicles (EVs) released from cells attract interest for their possible role in health and diseases. The detection and characterization of EVs is challenging due to the lack of specialized methodologies. Raman spectroscopy, however, has been suggested as a novel approach for biochemical analysis of EVs. To extract information from the spectra, a novel deep learning architecture is explored as a versatile variant of autoencoders. The proposed architecture considers the frequency range separately from the intensity of the spectra. This enables the model to adapt to the frequency range, rather than requiring that all spectra be pre-processed to the same frequency range as it was trained on. It is demonstrated that the proposed architecture accepts Raman spectra of EVs and lipoproteins from 13 biological sources and from two laboratories. High reconstruction accuracy is maintained despite large variances in frequency range and noise level. It is also shown that the architecture is able to cluster the biological nanoparticles by their Raman spectra and differentiate them by their origin without pre-processing of the spectra or supervision during learning. The model performs label-free differentiation, including separating EVs from activated vs. non-activated blood platelets and EVs/lipoproteins from prostate cancer patients versus non-cancer controls. The differentiation is evaluated by creating a neural network classifier that observes the features extracted by the model to classify the spectra according to their sample origin. The classification reveals a test sensitivity of 92.2 % and selectivity of 92.3 % over 769 measurements from two labs that have different measurement configurations.

Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity.

BACKGROUND: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism. OBJECTIVES: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets. METHODS: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied. RESULTS: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold. CONCLUSION: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.

Demonstrating low Raman background in UV-written SiO2 waveguides.

Raman spectroscopy can give a chemical 'fingerprint' from both inorganic and organic samples, and has become a viable method of measuring the chemical composition of single biological particles. In parallel, integration of waveguides and microfluidics allows for the creation of miniaturized optical sensors in lab-on-a-chip devices. The prospect of combining integrated optics and Raman spectroscopy for Raman-on-chip offers new opportunities for optical sensing. A major limitation for this is the Raman background of the waveguide. This background is very low for optical fibers but remains a challenge for planar waveguides. In this work, we demonstrate that UV-written SiO2 waveguides, designed to mimic the performance of optical fibers, offer a significantly lower background than competing waveguide materials such as Si3N4. The Raman scattering in the waveguides is measured in absolute units and compared to that of optical fibers and Si3N4 waveguides. A limited study of the sensitivity of the Raman scattering to changes in pump wavelength and in waveguide design is also conducted. It is revealed that UV-written SiO2 waveguides offer a Raman background lower than -107.4 dB relative to a 785 nm pump and -106.5 dB relative to a 660 nm pump. Furthermore, the UV-written SiO2 waveguide demonstrates a 15 dB lower Raman background than a Si3N4 waveguide and is only 8.7 - 10.3 dB higher than optical fibers. Comparison with a polystyrene bead (in free space, diameter 7 µm) reveal an achievable peak SNR of 10.4 dB, showing the potential of UV-SiO2 as a platform for a Raman-on-chip device capable of measuring single particles.

Metasurface supporting quasi-BIC for optical trapping and Raman-spectroscopy of biological nanoparticles.

Optical trapping combined with Raman spectroscopy have opened new possibilities for analyzing biological nanoparticles. Conventional optical tweezers have proven successful for trapping of a single or a few particles. However, the method is slow and cannot be used for the smallest particles. Thus, it is not adapted to analyze a large number of nanoparticles, which is necessary to get statistically valid data. Here, we propose quasi-bound states in the continuum (quasi-BICs) in a silicon nitride (Si3N4) metasurface to trap smaller particles and many simultaneously. The quasi-BIC metasurface contains multiple zones with high field-enhancement ('hotspots') at a wavelength of 785 nm, where a single nanoparticle can be trapped at each hotspot. We numerically investigate the optical trapping of a type of biological nanoparticles, namely extracellular vesicles (EVs), and study how their presence influences the resonance behavior of the quasi-BIC. It is found that perturbation theory and a semi-analytical expression give good estimates for the resonance wavelength and minimum of the potential well, as a function of the particle radius. This wavelength is slightly shifted relative to the resonance of the metasurface without trapped particles. The simulations show that the Q-factor can be increased by using a thin metasurface. The thickness of the layer and the asymmetry of the unit cell can thus be used to get a high Q-factor. Our findings show the tight fabrication tolerances necessary to make the metasurface. If these can be overcome, the proposed metasurface can be used for a lab-on-a-chip for mass-analysis of biological nanoparticles.

Dielectric optical nanoantennas.

Nanophotonics allows the manipulation of light on the subwavelength scale. Optical nanoantennas are nanoscale elements that enable increased resolution in bioimaging, novel photon sources, solar cells with higher absorption, and the detection of fluorescence from a single molecule. While plasmonic nanoantennas have been extensively explored in the literature, dielectric nanoantennas have several advantages over their plasmonic counterparts, including low dissipative losses and near-field enhancement of both electric and magnetic fields. Nanoantennas increase the optical density of states, which increase the rate of spontaneous emission due to the Purcell effect. The increase is quantified by the Purcell factor, which depends on the mode volume and the quality factor. It is one of the main performance parameters for nanoantennas. One particularly interesting feature of dielectric nanoantennas is the possibility of integrating them into optical resonators with a high quality-factor, further improving the performance of the nanoantennas and giving very high Purcell factors. This review introduces the properties and parameters of dielectric optical nanoantennas, and gives a classification of the nanoantennas based on the number and shape of the nanoantenna elements. An overview of recent progress in the field is provided, and a simulation is included as an example. The simulated nanoantenna, a dimer consisting of two silicon nanospheres separated by a gap, is shown to have a very small mode volume, but a low quality-factor. Some recent works on photonic crystal resonators are reviewed, including one that includes a nanoantenna in the bowtie unit-cell. This results in an enormous increase in the calculated Purcell factor, from 200 for the example dimer, to 8 × 106 for the photonic crystal resonator. Some applications of dielectric nanoantennas are described. With current progress in the field, it is expected that the number of applications will grow and that nanoantennas will be incorporated into new commercial products. A list of relevant materials with high refractive indexes and low losses is presented and discussed. Finally, prospects and major challenges for dielectric nanoantennas are addressed.

Optical pressure and numerical simulation of optical forces.

Optical forces on a particle can be calculated using numerical methods and by integrating the Maxwell stress tensor over the surface of the particle. It is shown that this gives considerable numerical noise for the radiation force on particles with refractive index close to that of the surrounding medium and that a large number of mesh elements are necessary. It is found preferable to calculate the force from the local optical pressure, as this gives considerably less noise and requires significantly fewer mesh elements. Results are also compared with an analytical model based on Mie theory.

Rib waveguides for trapping and transport of particles.

Rib waveguides are investigated as an alternative to strip waveguides for planar trapping and transport of microparticles. Microparticles are successfully propelled along the surface of rib waveguides and trapped in the gap between opposing rib waveguides. The trapping capabilities of waveguide end facets formed by a single and opposing waveguide geometries are investigated. The slab beneath a rib waveguide continues to guide light after the end facet of a rib waveguide. Thus particles can be trapped in wider gaps formed by opposing rib waveguides than with strip waveguides. Rib waveguides were found more efficient in trapping a collection of particles in the gap and particles could be moved to different locations in the gap by changing the relative power in the two opposing rib waveguides. Numerical simulations are used to show that the trapping efficiency on the surface of rib and strip waveguides is comparable. The simulations also confirm the advantage of opposing rib waveguides for trapping particles in wide gaps. The low sidewalls of rib waveguides give low propagation losses and make it easy to integrate rib waveguides with other functions in a lab-on-a-chip where particle trapping and transport is required.

Sensitive on-chip methane detection with a cryptophane-A cladded Mach-Zehnder interferometer.

We report a methane sensor based on an integrated Mach-Zehnder interferometer, which is cladded by a styrene-acrylonitrile film incorporating cryptophane-A. Cryptophane-A is a supramolecular compound able to selectively trap methane, and its presence in the cladding leads to a 17-fold sensitivity enhancement. Our approach, based on 3 cm-long low-loss Si3N4 rib waveguides, results in a detection limit as low as 17 ppm. This is 1-2 orders of magnitude lower than typically achieved with chip-scale low-cost sensors.

On-chip phase measurement for microparticles trapped on a waveguide.

Polystyrene microparticles are trapped on a waveguide Young interferometer and the phase change caused by the trapped particles is measured. This is a novel, on-chip method that can be used to count and characterize trapped particles. The trapping of single particles is clearly identified. Simulations show that the phase change increases with the diameter up to 7 µm, while for larger particles, morphology-dependent resonances appear. For 7 µm particles, a phase change of -0.13 rad is measured, while the simulated value is -0.28 rad. Extensive simulations are carried out regarding the phase change, waveguide transmission and the forces on the particles, and also regarding sources of the discrepancy between simulations and measurements.

Optical transport, lifting and trapping of micro-particles by planar waveguides.

Optical waveguides can be used to trap and transport micro-particles. 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.

Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage.

Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

Serial Raman spectroscopy of particles trapped on a waveguide.

We demonstrate that Raman spectroscopy can be used to characterize and identify particles that are trapped and propelled along optical waveguides. To accomplish this, microscopic particles on a waveguide are moved along the waveguide and then individually addressed by a focused laser beam to obtain their characteristic Raman signature within 1 second acquisition time. The spectrum is used to distinguish between glass and polystyrene particles. After the characterization, the particles continue to be propelled along the straight waveguide. Alternatively, a waveguide loop with a gap is also investigated, and in this case particles are held in the gap for characterization before they are released.

Surface transport and stable trapping of particles and cells by an optical waveguide loop.

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.

Waveguide trapping of hollow glass spheres.

Microparticles can be trapped and propelled by the evanescent field of optical waveguides. As the evanescent field only stretches 100-200 nm from the surface of the waveguide, only the lower caps of the microparticles interact directly with the field. This is taken advantage of by trapping hollow glass spheres on waveguides in the same way as solid glass spheres. For the chosen waveguide, numerical simulations show that hollow microspheres with a shell thickness above 60 nm can be stably trapped, while spheres with thinner shells are repelled. The average refractive index of the sphere-field intersection volume is used to explain the result in a qualitative way.

Optical trapping and propulsion of red blood cells on waveguide surfaces.

We have studied optical trapping and propulsion of red blood cells in the evanescent field of optical waveguides. Cell propulsion is found to be highly dependent on the biological medium and serum proteins the cells are submerged in. Waveguides made of tantalum pentoxide are shown to be efficient for cell propulsion. An optical propulsion velocity of up to 6 µm/s on a waveguide with a width of ~6 µm is reported. Stable optical trapping and propulsion of cells during transverse flow is also reported.

Dielectric microsphere manipulation and chain assembly by counter-propagating waves in a channel waveguide.

We study the formation and the propulsion properties of chains of dielectric microspheres in the evanescent field of a channel waveguide made by Cs(+) ion-exchange. Particle chains are shown to move faster than single particles. We exploit counter-propagating waves for axial positioning of single and chains of microspheres. The particles can be propelled back and forth at will, and trapped at a given point for several minutes. We demonstrate that this technique can also be used to assemble a long, one-particle wide, chain.