Super-resolved multispectral lensless microscopy via angle-tilted, wavelength-multiplexed ptychographic modulation.

We report an angle-tilted, wavelength-multiplexed ptychographic modulation approach for multispectral lensless on-chip microscopy. In this approach, we illuminate the specimen with lights at five wavelengths simultaneously. A prism is added at the illumination path for spectral dispersion. Thus, lightwaves at different wavelengths hit the specimen at slightly different incident angles, breaking the ambiguities in mixed-state ptychographic reconstruction. At the detection path, we place a thin diffuser between the specimen and the monochromatic image sensor for encoding the spectral information into 2D intensity measurements. By scanning the sample to different x-y positions, we acquire a sequence of monochromatic images for reconstructing the five complex object profiles at the five wavelengths. An up-sampling procedure is integrated into the recovery process to bypass the resolution limit imposed by the imager pixel size. We demonstrate a half-pitch resolution of 0.55 µm using an image sensor with 1.85 µm pixel size. We also demonstrate quantitative and high-quality multispectral reconstructions of stained tissue sections for digital pathology applications.

Super-resolution microscopy via ptychographic structured modulation of a diffuser.

We report a new coherent imaging technique, termed ptychographic structured modulation (PSM), for quantitative super-resolution microscopy. In this technique, we place a thin diffuser (i.e., a scattering lens) in between the sample and the objective lens to modulate the complex light waves from the object. The otherwise inaccessible high-resolution object information can thus be encoded into the captured images. We then employ a ptychographic phase retrieval process to jointly recover the exit wavefront of the complex object and the unknown diffuser profile. Unlike the illumination-based super-resolution approach, the recovered image of our approach depends upon how the complex wavefront exits the sample-not enters it. Therefore, the sample thickness becomes irrelevant during reconstruction. After recovery, we can propagate the super-resolution complex wavefront to any position along the optical axis. We validate our approach using a resolution target, a quantitative phase target, a two-layer sample, and a thick polydimethylsiloxane sample. We demonstrate a 4.5-fold resolution gain over the diffraction limit. We also show that a four-fold resolution gain can be achieved with as few as ∼30 images. The reported approach may provide a quantitative super-resolution strategy for coherent light, x-ray, and electron imaging.

Axially shifted pattern illumination for macroscale turbidity suppression and virtual volumetric confocal imaging without axial scanning.

Structured illumination has been widely used for optical sectioning and 3D surface recovery. In a typical implementation, multiple images under non-uniform pattern illumination are used to recover a single object section. Axial scanning of the sample or the objective lens is needed for acquiring the 3D volumetric data. Here we demonstrate the use of axially shifted pattern illumination for virtual volumetric confocal imaging without axial scanning. In the reported approach, we project illumination patterns at a tilted angle with respect to the detection optics. As such, the illumination patterns shift laterally at different z sections, and the 3D sample information can be recovered based on the captured 2D images. We demonstrate the reported approach for virtual confocal imaging through a diffusing layer and underwater 3D imaging through diluted milk. We show that we can acquire the entire confocal volume in ∼1 s with a throughput of 420 megapixels per second. Our approach may provide new insights for developing confocal light ranging and detection systems in degraded visual environments.

Biocompatible Cantilevers for Mechanical Characterization of Zebrafish Embryos using Image Analysis.

We have developed a force sensing system to continuously evaluate the mechanical elasticity of micrometer-scale (a few hundred micrometers to a millimeter) live tissues. The sensing is achieved by measuring the deflection of force sensitive cantilevers through microscopic image analysis, which does not require electrical strain gauges. Cantilevers made of biocompatible polydimethylsiloxane (PDMS) were actuated by a piezoelectric actuator and functioned as a pair of chopsticks to measure the stiffness of the specimen. The dimensions of the cantilevers were easily adjusted to match the size, range, and stiffness of the zebrafish samples. In this paper, we demonstrated the versatility of this technique by measuring the mechanical elasticity of zebrafish embryos at different stages of development. The stiffness of zebrafish embryos was measured once per hour for 9 h. From the experimental results, we successfully quantified the stiffness change of zebrafish embryos during embryonic development.

A Review of Cerebral Shunts, Current Technologies, and Future Endeavors.

Objective. The use of cerebrospinal shunts is the standard of care for hydrocephalus. However, shunts are extremely vulnerable to failure and lack noninvasive methods to monitor their viability. We review current shunt technologies and attempts to improve their function. Methods. A PubMed search was performed to find literature on shunts and shunt function. Company brochures and websites were also used. Results. Fixed and variable pressure valves from four major companies are discussed. Also reviewed are siphon resistive devices, intracranial pressure sensors, and recent attempts on the development of cerebrospinal fluid sensors, including a micromechanical flow sensor we have recently developed. Conclusions. While variable pressure valves and siphon resistive devices have both had considerable success in dealing with variable intracranial pressure, a more sophisticated, continuous monitoring system is needed to ensure shunt viability and patient safety. An integrated flow sensor may provide the ability to track fluid flow and determine shunt functionality.

Angular light modulator using optical blinds.

Spatial light modulator (SLM) is widely used in imaging applications for modulating light intensity and phase delay. In this paper, we report a novel device concept termed angular light modulator (ALM). Different from the SLM, the reported ALM employs a tunable blind structure to modulate the angular components of the incoming light waves. For spatial-domain light modulation, the ALM can be directly placed in front of an image sensor for selecting different angular light components. In this case, we can sweep the slat angle of the blind structure and capture multiple images corresponding to different perspectives. These images can then be back-projected for 3D tomographic refocusing. By using a fixed slat angle, we can also convert the incident-angle information into intensity variations for wavefront sensing or introduce a translational shift to the defocused object for high-speed autofocusing. For Fourier-domain light modulation, the ALM can be placed at the pupil plane of an optical system for reinforcing the light propagating trajectories. We show that a pupil-plane-modulated system is able to achieve a better resolution for out-of-focus objects while maintaining the same resolution for in-focus objects. The reported ALM can be fabricated on the chip level and controlled by an external magnetic field. It may provide new insights for developing novel imaging and vision devices.

Microfluidic immunodetection of cancer cells via site-specific microcontact printing of antibodies on nanoporous surface.

We demonstrate an efficient method for cancer cell capture via cell line-specific protein deposition on nanoporous surface in microfluidic channels. Specifically, anti-epithelial cell adhesion molecules (EpCAM) were microcontact printed onto nanoporous silica substrate with optimal pore size of 4 nm, porosity of 52.4 ± 0.2%, and thin film thickness of 130 ± 0.5 nm. SkBr3, Colo205, and MDA-MB-435 cancer cells suspended in buffer solution were captured on the stamped nanoporous silica substrate. The method demonstrated significantly higher numbers of captured EpCAM-positive cancer cells within anti-EpCAM stamped areas. For site-selective cell capture, grooved microfluidic channels were designed to investigate effects of local confinement due to the laminar flows. Both theoretical modeling and experiments show that the integration of the microfluidic channels greatly enhances cell capture. Patterning of anti-EpCAM in areas of downward flow (optimal regions for cell capture), generated by grooves of the microchannel, enables higher capture numbers than that of stamped areas of upward flow (non-optimal). Fluorescence microscopy images were acquired for captured SkBr3 and Colo205 cells using anti-EpCAM on the nanoporous silica substrate. It was shown that higher numbers of cells were captured across all EpCAM-positive cell lines in optimal areas versus non-optimal areas. Spatial control and large scale patterning of proteins enable novel designs and productions of cost effective, high throughput, and integrated detection and analysis systems. Site-selective detection provides the capability of defining optimal locations for cell capture based on various channel geometries and flow profile. The demonstrated method shows great potential for point-of-care cancer diagnostic tools to quantify the progression and status of the disease.

Optical Elastography for Micropressure Characterization of Zebrafish Embryonic Cardiac Development.

The proper formation of the vertebrate embryonic heart relies on various mechanical forces which determine its form and function. Measuring these forces at the microscale of the embryo is a challenge. We propose a new tool utilizing high-resolution optical elastography and stiffness measurements of surrounding tissues to non-invasively track the changes in the pressure exerted by the heart on the neighboring yolk, as well as changes in contractile patterns during early cardiac growth in-vivo, using the zebrafish embryo as a model system. Cardiac development was characterized every three hours from 24 hours post-fertilization (hpf) to 30 hpf and compared between wildtype fish and those treated with MS-222, a commonly used fish anesthetic that decreases cardiac contractility. Wildtype embryos from 24 to 30 hpf showed an average yolk indentation pressure of 0.32 mmHg to 0.41 mmHg, respectively. MS-222 treated embryos showed an average yolk indentation pressure of 0.22 mmHg to 0.29 mmHg. Yolk indentation pressure between control and treated embryos at 24 hpf and 30 hpf showed a significant difference (p < 0.05). Our method allowed for contractility and pressure evaluation at these early developmental stages, which have not been previously reported in published literature, regardless of sample or imaging modality. This research could lead to a better understanding of heart development and improved diagnostic tools for congenital heart disease.

Lightsheet microscopy integrates single-cell optical visco-elastography and fluorescence cytometry of 3D live tissues.

Most common cytometry methods, including flow cytometry, observe suspended or fixed cells and cannot evaluate their structural roles in 3D tissues. However, cellular physical interactions are critical in physiological, developmental, and pathological processes. Here, we present a novel optical visco-elastography that characterizes single-cellular physical interactions by applying in-situ micro-mechanical perturbation to live microtissues under 3D lightsheet microscopy. The 4D digital image correlation (DIC) analysis of ~20,000 nodes tracked the compressive deformation of 3D tissues containing ~500 cells. The computational 3D image segmentation allowed cell-by-cell qualitative observation and statistical analysis, directly correlating multi-channel fluorescence and viscoelasticity. To represent epithelia-stroma interactions, we used a 3D organoid model of maternal-fetal interface and visualized solid-like, well-aligned displacement and liquid-like random motion between individual cells. The statistical analysis through our unique cytometry confirmed that endometrial stromal fibroblasts stiffen in response to decidualization. Moreover, we demonstrated in the 3D model that interaction with placental extravillous trophoblasts partially reverses the attained stiffness, which was supported by the gene expression analysis. Placentation shares critical cellular and molecular significance with various fundamental biological events such as cancer metastasis, wound healing, and gastrulation. Our analysis confirmed existing beliefs and discovered new insights, proving the broad applicability of our method.

Tunable directive radiation of surface-plasmon diffraction gratings.

We experimentally demonstrate tunable radiation from a periodic array of plasmonic nanoscatterers, tailored to convert surface plasmon polaritons into directive leaky modes. Extending our previous studies on efficient directional beaming based on leaky-wave radiation from periodic gratings driven by a subwavelength slit, we experimentally show dynamic beam sweeping by tuning the directional leaky-wave mechanism in real-time. Two alternative tuning mechanisms, wavelength- and index-mediated beam sweeping, are employed to modify the relative phase of scattered light at each grating edge and provide the required modification of the radiation angle.

Immunomagnetic nanoscreening of circulating tumor cells with a motion controlled microfluidic system.

Combining the power of immunomagnetic assay and microfluidic microchip operations, we successfully detected rare CTCs from clinical blood samples. The microfluidic system is operated in a flip-flop mode, where a computer-controlled rotational holder with an array of microfluidic chips inverts the microchannels. We have demonstrated both theoretically and experimentally that the direction of red blood cell (RBC) sedimentation with regards to the magnetic force required for cell separation is important for capture efficiency, throughput, and purity. The flip-flop operation reduces the stagnation of RBCs and non-specific binding on the capture surface by alternating the direction of the magnetic field with respect to gravity. The developed immunomagnetic microchip-based screening system exhibits high capture rates (more than 90%) for SkBr3, PC3, and Colo205 cell lines in spiked screening experiments and successfully isolates CTCs from patient blood samples. The proposed motion controlled microchip-based immunomagnetic system shows great promise as a clinical tool for cancer diagnosis and prognosis.

Light microscopy-based elastography for the mechanical characterization of zebrafish somitogenesis.

We evaluated the elasticity of live tissues of zebrafish embryos using label-free optical elastography. We employed a pair of custom-built elastic microcantilevers to gently compress a zebrafish embryo and used optical-tracking analysis to obtain the induced internal strain. We then built a finite element method (FEM) model and matched the strain with the optical analysis. The elastic moduli were found by minimizing the root-mean-square errors between the optical and FEM analyses. We evaluated the average elastic moduli of a developing somite, the overlying ectoderm, and the underlying yolk of seven zebrafish embryos during the early somitogenesis stages. The estimation results showed that the average elastic modulus of the somite increased from 150 to 700 Pa between 4- and 8-somite stages, while those of the ectoderm and the yolk stayed between 100 and 200 Pa, and they did not show significant changes. The result matches well with the developmental process of somitogenesis reported in the literature. This is among the first attempts to quantify spatially-resolved elasticity of embryonic tissues from optical elastography.

Electron microscopy imaging and mechanical characterization of T47D multicellular tumor spheroids-Older spheroids reduce interstitial space and become stiffer.

Multicellular cancer spheroids are an in vitro tissue model that mimics the three-dimensional microenvironment. As spheroids grow, they develop the gradients of oxygen, nutrients, and catabolites, affecting crucial tumor characteristics such as proliferation and treatment responses. The measurement of spheroid stiffness provides a quantitative measure to evaluate such structural changes over time. In this report, we measured the stiffness of size-matched day 5 and day 20 tumor spheroids using a custom-built microscale force sensor and conducted transmission electron microscopy (TEM) imaging to compare the internal structures. We found that older spheroids reduce interstitial spaces in the core region and became significantly stiffer. The measured elastic moduli were 260±100 and 680±150 Pa, for day 5 and day 20 spheroids, respectively. The day 20 spheroids showed an optically dark region in the center. Analyzing the high-resolution TEM images of spheroid middle sections across the diameter showed that the cells in the inner region of the day 20 spheroids are significantly larger and more closely packed than those in the outer regions. On the other hand, the day 5 spheroids did not show a significant difference between the inner and outer regions. The observed reduction of the interstitial space may be one factor that contributes to stiffer older spheroids.

Computational analysis of microfluidic immunomagnetic rare cell separation from a particulate blood flow.

We describe a computational analysis method to evaluate the efficacy of immunomagnetic rare cell separation from non-Newtonian particulate blood flow. The core procedure proposed here is calculation of local viscosity distributions induced by red blood cell (RBC) sedimentation. Numerical calculation methods have previously been introduced to simulate particulate behavior of individual RBCs. However, due to the limitation of the computational power, those studies are typically capable of calculating only a very small number (less than 100) of RBCs and are not suitable to analyze many practical separation methods for rare cells such as circulating tumor cells (CTCs). We introduce a sedimentation and viscosity model based on our experimental measurements. The computational field is divided into small unit control volumes, where the local viscosity distribution is dynamically calculated based on the experimentally found sedimentation model. For analysis of rare cell separation, the local viscosity distribution is calculated as a function of the volume RBC rate. The direction of gravity has an important role in such a sedimentation-involved cell separation system. We evaluated the separation efficacy with multiple design parameters including the channel design, channel operational orientations (inverted and upright), and flow rates. The results showed excellent agreement with real experiments to demonstrate the effectiveness of our computational analytical method. We demonstrated higher capture efficiency with the inverted microchannel configuration.We conclude that proper direction of blood sedimentation significantly enhances separation efficiency in microfluidic devices.

Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization.

This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (µTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 µl/sample using the µTweezers vs. ~157 µl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred µm-1500 µm) 3D cell cultures. The µTweezers applied and measured nano-millinewton forces through cantilever' deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids' applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young's modulus of the control group (Control-day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The µTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators.

Label-Free Morphological Phenotyping of In Vitro 3D Microtumors.

By combining novel micro-scale manipulation cantilevers with commercially available, widely used 3D light microscopy, we were able to develop a new method of 3D elastography specialized for the analysis of 3D microtumors. Existing mechanical characterization methods are available for the study of single cells, using forces in the range of sub pN to a few hundred nN, or of larger tissues, with forces greater than 1 mN. Our method supports the mechanical analysis of micro- to meso-scale 3D tissues, such as multicellular spheroids (200-300 µm diameter), by applying forces in the range of sub-hundred nN to sub-mN, while also maintaining a spatial resolution of elasticity measurement as small as 20-30 µm. We use a differential interference contrast (DIC)/confocal microscope to obtain a 4D (x, y, z, and indentation steps) image sequence, which is then analyzed using our custom 3D pattern-tracking MATLAB program. With this method, we have been able to show structural and spatial heterogeneity among single cells and surrounding regions in tumor spheroids, and between different cell types in tumor-fibroblast co-cultured spheroids. Our method has the potential to both bridge the gap between in vitro monolayer culture systems and in vivo animal studies and add a mechanical component to existing biological assays.

Ptychographic sensor for large-scale lensless microbial monitoring with high spatiotemporal resolution.

Traditional microbial detection methods often rely on the overall property of microbial cultures and cannot resolve individual growth event at high spatiotemporal resolution. As a result, they require bacteria to grow to confluence and then interpret the results. Here, we demonstrate the application of an integrated ptychographic sensor for lensless cytometric analysis of microbial cultures over a large scale and with high spatiotemporal resolution. The reported device can be placed within a regular incubator or used as a standalone incubating unit for long-term microbial monitoring. For longitudinal study where massive data are acquired at sequential time points, we report a new temporal-similarity constraint to increase the temporal resolution of ptychographic reconstruction by 7-fold. With this strategy, the reported device achieves a centimeter-scale field of view, a half-pitch spatial resolution of 488 nm, and a temporal resolution of 15-s intervals. For the first time, we report the direct observation of bacterial growth in a 15-s interval by tracking the phase wraps of the recovered images, with high phase sensitivity like that in interferometric measurements. We also characterize cell growth via longitudinal dry mass measurement and perform rapid bacterial detection at low concentrations. For drug-screening application, we demonstrate proof-of-concept antibiotic susceptibility testing and perform single-cell analysis of antibiotic-induced filamentation. The combination of high phase sensitivity, high spatiotemporal resolution, and large field of view is unique among existing microscopy techniques. As a quantitative and miniaturized platform, it can improve studies with microorganisms and other biospecimens at resource-limited settings.

Light focusing by slot Fabry-Perot photonic crystal nanoresonator on scanning tip.

We numerically investigate the propagation of light through the photonic crystal (PhC) waveguide on low refraction index material for near-field light focusing at the visible wavelength (635 nm) by incorporating a center air slot and Fabry-Perot resonator on the scanning tip. Perturbations by water and substrate refraction index changes of the PhC are analyzed by the finite-difference time-domain method to show minimal impact on light confinement and throughput. Such a total dielectric probe tip design has great potential to complement the current widely used metal-coated optical-fiber-based light confinement probe.

Enhanced microcontact printing of proteins on nanoporous silica surface.

We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

Studies of nanoparticle delivery with in vitro bio-engineered microtissues.

A variety of engineered nanoparticles, including lipid nanoparticles, polymer nanoparticles, gold nanoparticles, and biomimetic nanoparticles, have been studied as delivery vehicles for biomedical applications. When assessing the efficacy of a nanoparticle-based delivery system, in vitro testing with a model delivery system is crucial because it allows for real-time, in situ quantitative transport analysis, which is often difficult with in vivo animal models. The advent of tissue engineering has offered methods to create experimental models that can closely mimic the 3D microenvironment in the human body. This review paper overviews the types of nanoparticle vehicles, their application areas, and the design strategies to improve delivery efficiency, followed by the uses of engineered microtissues and methods of analysis. In particular, this review highlights studies on multicellular spheroids and other 3D tissue engineering approaches for cancer drug development. The use of bio-engineered tissues can potentially provide low-cost, high-throughput, and quantitative experimental platforms for the development of nanoparticle-based delivery systems.