Use of high frequency electrorotation to identify cytoplasmic changes in cells non-disruptively.

In this paper we demonstrate how the use of frequencies ranging from 50 kHz to 5 GHz in the analysis of cells by electrorotation can open the path to the identification of differences not detectable by conventional set-ups. Earlier works usually reported electrorotation devices operating below 20 MHz, limiting the response obtained to properties associated with the cell membrane. Those devices are thus unable to resolve the physiological properties in the cytoplasm. We used microwave-based technology to extend the frequency operation to 5 GHz. At high frequencies (from tens of MHz to GHz), the electromagnetic signal passes through the membrane and allows probing the cytoplasm. This enables several applications, such as cell classification, and viability analysis. Additionally, the use of conventional microfabrication techniques reduces the cost and complexity of analysis, compared to other non-invasive methods. We demonstrated the potential of this set-up by identifying two different populations of T-lymphocytes not distinguishable through visual assessment. We also assessed the effect of calcein on cell cytoplasmic properties and used it as a controlled experiment to demonstrate the possibility of this method to detect changes happening predominantly in the cytoplasm.

Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation.

Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (Ct) distributions that can be fitted using a log-normal probability distribution. From the fit parameters, the amplification efficiency can be calculated. Template inaccessibility, on the other hand, leads to a different rain pattern, in which a distinct exponential tail in the Ct distribution can be observed. Using our model, it is possible to determine if the amplification efficiency, template accessibility, or another source is the main contributor of rain in dPCR assays. We envision that this model will facilitate and speed up dPCR assay optimization and provide an indication for the accuracy of the assay.

Rigorous analysis of the axial acoustic radiation force on a spherical object for single-beam acoustic tweezing applications.

Acoustic tweezers are increasingly utilized for the contactless manipulation of small particles. This paper provides a theoretical model demonstrating the acoustic manipulation capabilities of single-beam acoustic transducers. Analytical formulas are derived for the acoustic radiation force on an isotropic spherical object of arbitrary size, centered on a circular piston, simply supported and clamped radiator in an inviscid fluid. Using these results, the existence of a negative axial force pulling the object closer to the radiator is revealed and explored. These findings offer further insight into the feasibility of trapping objects in the near-field of a single-beam acoustic transducer. The calculations illustrate the trapping capabilities of the different emitters as a function of radiator size, particle size, and distance from the source and highlight the impact of radiator boundary conditions. Manipulation of a cell-like fluid sphere in water and an expanded polystyrene sphere in air are studied in more detail with results that are validated through finite element analysis. The developed theoretical model allows fast evaluation of acoustic radiation forces which could aid in the development of relatively simple and inexpensive contactless manipulation solutions.

Fast compressive lens-free tomography for 3D biological cell culture imaging.

We present a compressive lens-free technique that performs tomographic imaging across a cubic millimeter-scale volume from highly sparse data. Compared with existing lens-free 3D microscopy systems, our method requires an order of magnitude fewer multi-angle illuminations for tomographic reconstruction, leading to a compact, cost-effective and scanning-free setup with a reduced data acquisition time to enable high-throughput 3D imaging of dynamic biological processes. We apply a fast proximal gradient algorithm with composite regularization to address the ill-posed tomographic inverse problem. Using simulated data, we show that the proposed method can achieve a reconstruction speed ∼10× faster than the state-of-the-art inverse problem approach in 3D lens-free microscopy. We experimentally validate the effectiveness of our method by imaging a resolution test chart and polystyrene beads, demonstrating its capability to resolve micron-size features in both lateral and axial directions. Furthermore, tomographic reconstruction results of neuronspheres and intestinal organoids reveal the potential of this 3D imaging technique for high-resolution and high-throughput biological applications.

Pixel super-resolution for lens-free holographic microscopy using deep learning neural networks.

Lens-free holographic microscopy (LFHM) provides a cost-effective tool for large field-of-view imaging in various biomedical applications. However, due to the unit optical magnification, its spatial resolution is limited by the pixel size of the imager. Pixel super-resolution (PSR) technique tackles this problem by using a series of sub-pixel shifted low-resolution (LR) lens-free holograms to form the high-resolution (HR) hologram. Conventional iterative PSR methods require a large number of measurements and a time-consuming reconstruction process, limiting the throughput of LFHM in practice. Here we report a deep learning-based PSR approach to enhance the resolution of LFHM. Compared with the existing PSR methods, our neural network-based approach outputs the HR hologram in an end-to-end fashion and maintains consistency in resolution improvement with a reduced number of LR holograms. Moreover, by exploiting the resolution degradation model in the imaging process, the network can be trained with a data set synthesized from the LR hologram itself without resorting to the HR ground truth. We validated the effectiveness and the robustness of our method by imaging various types of samples using a single network trained on an entirely different data set. This deep learning-based PSR approach can significantly accelerate both the data acquisition and the HR hologram reconstruction processes, therefore providing a practical solution to fast, lens-free, super-resolution imaging.

Development and validation of a glass-silicon microdroplet-based system to measure sulfite concentrations in beverages.

Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5'-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstract ᅟ.

Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy.

Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.

Fast and robust Fourier domain-based classification for on-chip lens-free flow cytometry.

The development of portable haematology analysers receives increased attention due to their deployability in resource-limited or emergency settings. Lens-free in-line holographic microscopy is one of the technologies that is being pushed forward in this regard as it eliminates complex and expensive optics, making miniaturisation and integration with microfluidics possible. On-chip flow cytometry enables high-speed capturing of individual cells in suspension, giving rise to high-throughput cell counting and classification. To perform a real-time analysis on this high-throughput content, we propose a fast and robust framework for the classification of leukocytes. The raw data consists of holographic acquisitions of leukocytes, captured with a high-speed camera as they are flowing through a microfluidic chip. Three different types of leukocytes are considered: granulocytes, monocytes and T-lymphocytes. The proposed method bypasses the reconstruction of the holographic data altogether by extracting Zernike moments directly from the frequency domain. By doing so, we introduce robustness to translations and rotations of cells, as well as to changes in distance of a cell with respect to the image sensor, achieving classification accuracies up to 96.8%. Furthermore, the reduced computational complexity of this approach, compared to traditional frameworks that involve the reconstruction of the holographic data, allows for very fast processing and classification, making it applicable in high-throughput flow cytometry setups.

Full-wafer in-situ fabrication and packaging of microfluidic flow cytometer with photo-patternable adhesive polymers.

Integration of microelectronics with microfluidics enables sophisticated lab-on-a-chip devices for sensing and actuation. In this paper, we investigate a novel method for in-situ microfluidics fabrication and packaging on wafer level. Two novel photo-patternable adhesive polymers were tested and compared, PA-S500H and DXL-009. The microfluidics fabrication method employs photo lithographical patterning of spin coated polymer films of PA or DXL and direct bonding of formed microfluidics to a top glass cover using die-to-wafer level bonding. These new adhesive materials remove the need for additional gluing layers. With this approach, we fabricated disposable microfluidic flow cytometers and evaluated the performance of those materials in the context of this application. DXL-009 exhibits lower autofluorescence compared to PA-S500H which improves detection sensitivity of fluorescently stained cells. Results obtained from the cytotoxicity test reveals that both materials are biocompatible. The functionality of these materials was demonstrated by detection of immunostained monocytes in microfluidic flow cytometers. The flexible, fully CMOS compatible fabrication process of these photo-patternable adhesive materials will simplify prototyping and mass manufacturing of sophisticated microfluidic devices with integrated microelectronics.

All-Dielectric Antenna Wavelength Router with Bidirectional Scattering of Visible Light.

An optical antenna forms the subwavelength bridge between free space optical radiation and localized electromagnetic energy. Its localized electromagnetic modes strongly depend on its geometry and material composition. Here, we present the design and experimental realization of a novel V-shaped all-dielectric antenna based on high-index amorphous silicon with a strong magnetic dipole resonance in the visible range. As a result, it exhibits extraordinary bidirectional scattering into diametrically opposite directions. The scattering direction is effectively controlled by the incident wavelength, rendering the antenna a passive bidirectional wavelength router. A detailed multipole decomposition analysis reveals that the excitation and abrupt phase change of an out-of-plane polarized magnetic dipole and an in-plane electric quadrupole are essential for the directivity switching. Previously, noble metals have been extensively exploited for plasmonic directional nanoantenna design. However, these inevitably suffer from high intrinsic ohmic losses and a relatively weak magnetic response to the incident light. Compared to a similar gold plasmonic nanoantenna design, we show that the silicon-based antennas demonstrate stronger magnetic scattering with minimal absorption losses. Our results indicate that all-dielectric antennas will open exciting possibilities for efficient manipulation of light-matter interactions.

Photoresistance switching of plasmonic nanopores.

Fast and reversible modulation of ion flow through nanosized apertures is important for many nanofluidic applications, including sensing and separation systems. Here, we present the first demonstration of a reversible plasmon-controlled nanofluidic valve. We show that plasmonic nanopores (solid-state nanopores integrated with metal nanocavities) can be used as a fluidic switch upon optical excitation. We systematically investigate the effects of laser illumination of single plasmonic nanopores and experimentally demonstrate photoresistance switching where fluidic transport and ion flow are switched on or off. This is manifested as a large (∼ 1-2 orders of magnitude) increase in the ionic nanopore resistance and an accompanying current rectification upon illumination at high laser powers (tens of milliwatts). At lower laser powers, the resistance decreases monotonically with increasing power, followed by an abrupt transition to high resistances at a certain threshold power. A similar rapid transition, although at a lower threshold power, is observed when the power is instead swept from high to low power. This hysteretic behavior is found to be dependent on the rate of the power sweep. The photoresistance switching effect is attributed to plasmon-induced formation and growth of nanobubbles that reversibly block the ionic current through the nanopore from one side of the membrane. This explanation is corroborated by finite-element simulations of a nanobubble in the nanopore that show the switching and the rectification.

Mode parity-controlled Fano- and Lorentz-like line shapes arising in plasmonic nanorods.

We present the experimental observation of spectral lines of distinctly different shapes in the optical extinction cross-section of metallic nanorod antennas under near-normal plane wave illumination. Surface plasmon resonances of odd mode parity present Fano interference in the scattering cross-section, resulting in asymmetric spectral lines. Contrarily, modes with even parity appear as symmetric Lorentzian lines. Finite element simulations are used to verify the experimental results. The emergence of either constructive or destructive mode interference is explained with a semianalytical 1D line current model. This simple model directly explains the mode-parity dependence of the Fano-like interference. Plasmonic nanorods are widely used as half-wave optical dipole antennas. Our findings offer a perspective and theoretical framework for operating these antennas at higher-order modes.

Measuring mass of nanoparticles and viruses in liquids with nanometer-scale pores.

Nanopores have recently been developed for the detection and physical characterization of nanoparticles, viruses, proteins, nucleic acids, and other macromolecules in liquids. The method provides the ability to rapidly estimate the size and electrical charge of analytes over a wide range of concentration, potentially with small sample volumes and low cost. Here, we use the technique to measure the mass of nanoparticles and viruses and their sedimentation. The analyte sedimentation-time measurement provides an estimate for the nanoparticle mass-density. We also show that the method can be used with samples at low concentration and in small volumes.

Harnessing plasmon-induced ionic noise in metallic nanopores.

The ionic properties of a metal-coated silicon nanopore were examined in a nanofluidic system. We observed a strong increase of the ionic noise upon laser light illumination. The effect appeared to be strongly mediated by the resonant excitation of surface plasmons in the nanopore as was demonstrated by means of ionic mapping of the plasmonic electromagnetic field. Evidence from both simulations and experiments ruled out plasmonic heating as the main source of the noise, and point toward photoinduced electrochemical catalysis at the semiconductor-electrolyte interface. This ionic mapping technique described is opening up new opportunities on noninvasive applications ranging from biosensing to energy conversion.

Unidirectional side scattering of light by a single-element nanoantenna.

Unidirectional side scattering of light by a single-element plasmonic nanoantenna is demonstrated using full-field simulations and back focal plane measurements. We show that the phase and amplitude matching that occurs at the Fano interference between two localized surface plasmon modes in a V-shaped nanoparticle lies at the origin of this effect. A detailed analysis of the V-antenna modeled as a system of two coherent point-dipole sources elucidates the mechanisms that give rise to a tunable experimental directivity as large as 15 dB. The understanding of Fano-based directional scattering opens a way to develop new directional optical antennas for subwavelength color routing and self-referenced directional sensing. In addition, the directionality of these nanoantennas can increase the detection efficiency of fluorescence and surface enhanced Raman scattering.

On-chip flow cytometer using integrated photonics for the detection of human leukocytes.

Differentiation between leukocyte subtypes like monocytes and lymphocytes is essential for cell therapy and research applications. To guarantee the cost-effective delivery of functional cells in cell therapies, billions of cells must be processed in a limited time. Yet, the sorting rates of commercial cell sorters are not high enough to reach the required yield. Process parallelization by using multiple instruments increases variability and production cost. A compact solution with higher throughput can be provided by multichannel flow cytometers combining fluidics and optics on-chip. In this work, we present a micro-flow cytometer with monolithically integrated photonics and fluidics and demonstrate that both the illumination of cells, as well as the collection of scattered light, can be realized using photonic integrated circuits. Our device is the first with sufficient resolution for the discrimination of lymphocytes and monocytes. Innovations in microfabrication have enabled complete integration of miniaturized photonic components and fluidics in a CMOS-compatible wafer stack. In combination with external optics, the device is ready for the collection of fluorescence using the on-chip excitation.

Boosting the figure-of-merit of LSPR-based refractive index sensing by phase-sensitive measurements.

Localized surface plasmon resonances possess very interesting properties for a wide variety of sensing applications. In many of the existing applications, only the intensity of the reflected or transmitted signals is taken into account, while the phase information is ignored. At the center frequency of a (localized) surface plasmon resonance, the electron cloud makes the transition between in- and out-of-phase oscillation with respect to the incident wave. Here we show that this information can experimentally be extracted by performing phase-sensitive measurements, which result in linewidths that are almost 1 order of magnitude smaller than those for intensity based measurements. As this phase change is an intrinsic property of a plasmon resonance, this opens up many possibilities for boosting the figure-of-merit (FOM) of refractive index sensing by taking into account the phase of the plasmon resonance. We experimentally investigated this for two model systems: randomly distributed gold nanodisks and gold nanorings on top of a continuous gold layer and a dielectric spacer and observed FOM values up to 8.3 and 16.5 for the respective nanoparticles.

Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and multiplexed gene delivery.

Spatially resolved transfection, intracellular delivery of proteins and nucleic acids, has the potential to drastically speed up the discovery of biologically active cargos, for instance for the development of cell therapies or new genome engineering tools. We recently demonstrated the use of a high-density microelectrode array for the targeted electrotransfection of cells grown on its surface, a process called High-Definition Electroporation (HD-EP). We also developed a framework based on Design of Experiments to quickly establish optimized electroporation conditions across five different electrical pulse parameters. Here, we used this framework to optimize the transfection efficiency of primary fibroblasts with a mCherry-encoding mRNA, resulting in 98% of the cells expressing the desired fluorescent protein without any sign of cell death. That transfection yield is the highest reported so far for electroporation. Moreover, varying the pulse number was shown to modulate the fluorescence intensity of cells, indicating the dosage-controlled delivery of mRNA and protein expression. Finally, exploiting the single-electrode addressability of the microelectrode array, we demonstrated spatially resolved, high efficiency, sequential transfection of cells with three distinct mRNAs. Since the chip can be easily redesigned to feature a much large number of electrodes, we anticipate that this methodology will enable the development of dedicated screening platforms for analysis of mRNA variants at scale.

Electric field gradient focusing with electro-osmotic flow to reduce analyte dispersion: Concept and numerical investigation.

In proteomics, the need to precisely examine the protein compounds of small samples, requires sensitive analytical methods which can separate and enrich compounds with high precision. Current techniques require a minimal analysis time to obtain satisfactory compound separation where longer analysis time means better separation of compounds. But, molecular diffusion will create broadening of the separated compound bands over time, increasing the peak width, and thus reducing the resolution and the enrichment. Electric field gradient focusing (EFGF) is a separation technique, in which proteins are simultaneously separated and enriched by balancing a gradient electrostatic force with a constant hydrodynamic drag force. Because of this balance, analytes are continuously pushed back to their focusing point, limiting the time-dependent peak broadening due to molecular diffusion. Current EFGF techniques are however still suffering from peak broadening because of flow-profile inhomogeneities. In this paper, we propose to use AC electro-osmotic flow (AC EOF) to create a homogeneous flow in EFGF. The interference between the electric field gradient and the AC EOF was thoroughly analysed and the concept was validated using numerical simulations. The results show that a plug flow is obtained on top of a small, distorted boundary layer. While applying different DC electric fields in the electrolyte, a constant flow velocity can be obtained by including a DC offset to the electrodes generating the AC EOF. The plug flow is then maintained over the whole separation channel length, while an electric field gradient is applied. This way, the flow-induced contribution to peak broadening can be minimized in EFGF devices. By modelling the separation of green fluorescent protein (GFP) and R-Phycoerythrin (R-PE), it was shown that the peak width of separated compounds can be reduced and that the separation resolution can be improved, compared to current EFGF methods.

A Microfluidics Approach for Ovarian Cancer Immune Monitoring in an Outpatient Setting.

Among cancer diagnoses in women, ovarian cancer has the fifth-highest mortality rate. Current treatments are unsatisfactory, and new therapies are highly needed. Immunotherapies show great promise but have not reached their full potential in ovarian cancer patients. Implementation of an immune readout could offer better guidance and development of immunotherapies. However, immune profiling is often performed using a flow cytometer, which is bulky, complex, and expensive. This equipment is centralized and operated by highly trained personnel, making it cumbersome and time-consuming. We aim to develop a disposable microfluidic chip capable of performing an immune readout with the sensitivity needed to guide diagnostic decision making as close as possible to the patient. As a proof of concept of the fluidics module of this concept, acquisition of a limited immune panel based on CD45, CD8, programmed cell death protein 1 (PD1), and a live/dead marker was compared to a conventional flow cytometer (BD FACSymphony). Based on a dataset of peripheral blood mononuclear cells of 15 patients with ovarian cancer across different stages of treatment, we obtained a 99% correlation coefficient for the detection of CD8+PD1+ T cells relative to the total amount of CD45+ white blood cells. Upon further system development comprising further miniaturization of optics, this microfluidics chip could enable immune monitoring in an outpatient setting, facilitating rapid acquisition of data without the need for highly trained staff.