Although co-delivery of small-molecule drugs and siRNAs is a promising platform strategy for combination therapy, current delivery systems fail to achieve efficient endosomal escape, limiting cytosolic siRNA bioavailability and therapeutic efficacy. Conventional lipid nanoparticles (LNPs) can co-encapsulate chemotherapeutics and siRNA, but their poor endosomal escape results in suboptimal siRNA bioavailability. Here, we report a novel type of sphere-like nanoparticle (SNP) platform for co-delivery of siRNA and small-molecule drugs that overcomes these limitations. In this system, doxorubicin (DOX) is intercalated within a Janus base nanotube (JBNt) structure, while siRNA is encapsulated through electrostatic interactions, enabling stable co-packaging. Notably, SNPs exhibit significantly enhanced endosomal escape compared with lipid nanoparticles, leveraging JBNt's endosomal escape, attributable to its distinct proton-sponge-mediated buffer capacity, consequently promoting efficient, coordinated cytosolic delivery of both cargos. In a proof-of-concept study, SNP-mediated co-delivery of Multidrug Resistance 1 gene (MDR1/ABCB1)-targeting siRNA and DOX was associated with effective gene silencing and enhanced apoptosis in cancer cells, tumor spheroids, and murine ovarian tumor xenograft models. Collectively, these findings deliver proof-of-concept evidence for SNPs as a promising co-delivery platform for RNA and chemotherapeutics to overcome chemoresistance and improve anticancer efficacy.

Understanding the multiscale mechanics of the colorectum is essential for uncovering the mechanotransductive pathways underlying visceral nociception. Intraluminal distension of the large intestine reliably evokes pain in disorders of gut-brain interaction (DGBIs), yet the tissue-level and nerve fiber-level responses to mechanical loading remain poorly defined. Here, we present results from our new biomechanical testing framework that integrates uniaxial circumferential extension with high-resolution optical imaging to quantify deformation in both bulk colorectal tissue and embedded sensory nerve fibers. We tested intact, cylindrical colorectal segments from mice using intraluminal stainless-steel rods to apply circumferential stretch while maintaining a planar imaging field. We measured bulk-tissue deformation via Digital Image Correlation (DIC), while we assessed stretch in nerve fibers through fluorescence imaging of VGLUT2-labeled afferents analyzed using custom fiber-network analyses. We tested our null hypothesis that the Poisson's function was greater than zero, i.e. νGreen>0. Across all specimens, the average median νGreen was -0.842, with regional values of -0.539, -0.663, and -1.322 for the colonic, intermediate, and rectal segments, respectively. Using a one-sided, one-sample t-test with a significance level (α) of 0.05, the total sample size (n=30) achieves >99.99% power to reject the null hypothesis. Regional analyses (n=10 per region) achieve powers of 89.66%, 96.73%, and 99.55% for the colonic, intermediate, and rectal regions, respectively, to reject the null hypothesis. Corresponding analyses of the nerve fibers across nine specimens revealed an average median stretch ratio of 1.063, indicating approximately six percent elongation, with substantial heterogeneity driven by fiber orientation. Our results confirm that the colorectum exhibits auxetic mechanics, and those mechanics transmit to embedded sensory nerve fibers, thus enhancing our understanding of mechanotransduction (e.g. mechanical and nociceptive signaling) and informing both development of treatment targets for visceral pain and design of bioinspired auxetic materials.

We introduce a 3D mechanically adaptive viscoelastic cell-network model that links single-cell interactions to emergent tissue rheology. Unlike existing continuum or cell-based models, viscoelasticity is embedded within discrete, mechanically adaptive intercellular connections, allowing tissue-scale rheology and phenomena such as swirling and jamming to arise from single-cell behaviors and connection remodeling. The framework is motivated by recent advances in three-dimensional imaging and structural analysis that resolve single-cell behaviors within aggregates. It is validated against two gold-standard bulk assays performed on spherical aggregates: micropipette aspiration and Hertzian plate compression. Under aspiration, the model demonstrates a transition from elastic deformation to viscous creep governed by localized packing and emergent jamming at the aspirated neck, accompanied by increased mechanically adaptive remodeling. Under compression, core rheology determines deformation mode: liquid-like aggregates exhibit enhanced swirling, consistent with experimental observations, whereas solid-like aggregates exhibit affine, Poisson-like deformation. These results bridge cell-scale dynamics and quantifiable tissue rheology including elastic modulus and vicosity, providing a framework to interpret emerging 3D measurements of multicellular mechanics.

Transitions of biological tissues between solid-like and liquid-like phases have been of great recent interest. Here, the first successful cell-by-cell evaluation of tissue viscoelastic transition is presented. An in situ micro-mechanical perturbation is applied to a microtissue, and the resulting volumetric deformation is evaluated using 3D light-sheet microscopy and digital image correlation (DIC), quantifying both solid-like, well-aligned displacement and liquid-like swirling motion between individual cells. The viscoelastic transition of fibroblasts is crucial in fundamental physiological events, such as placentation, cancer dissemination, and wound healing. This study investigates 3D organoid systems modeling maternal-fetal and tumor-stroma interfaces, demonstrating established molecular and structural parallels. The analysis visualizes individual cells in stromal-epithelial interactions and how they collectively alter tissue viscoelastic properties. It also enables in-silico microdissection, linking single-cell viscoelasticity with multi-channel fluorescence. RNAseq analysis of endometrial stromal fibroblasts shows that decidualization activates mechano-transcriptional regulators, including myocardin-related transcription factors (MRTFs), associated with increased cellular contractility and actomyosin mobilization. Knocking down MRTFA in cancer-associated fibroblasts in the tumor-fibroblast co-culture 3D model induces significant changes in fibroblast properties, mirroring those observed in the maternal-fetal interface model, highlighting parallels between placentation and cancer invasion. This analysis confirms existing beliefs and discovers new insights broadly applicable to studying organoids, embryos, tumors, and other tissues.

Synthetic aperture imaging has enabled breakthrough observations from radar to astronomy. However, optical implementation remains challenging due to stringent wavefield synchronization requirements among multiple receivers. Here we present the multiscale aperture synthesis imager (MASI), which utilizes parallelism to break complex optical challenges into tractable sub-problems. MASI employs a distributed array of coded sensors that operate independently yet coherently to surpass the diffraction limit of single receiver. It combines the propagated wavefields from individual sensors through a computational phase synchronization scheme, eliminating the need for overlapping measurement regions to establish phase coherence. Light diffraction in MASI naturally expands the imaging field, generating phase-contrast visualizations that are substantially larger than sensor dimensions. Without using lenses, MASI resolves sub-micron features at ultralong working distances and reconstructs 3D shapes over centimeter-scale fields. MASI transforms the intractable optical synchronization problem into a computational one, enabling practical deployment of scalable synthetic aperture systems at optical wavelengths.

In this study, we examined the effects of gold nanoparticles (AuNPs) and ultrastructural alterations on breast cancer cell line (T47D) spheroid. Tissue morphological changes, AuNP interactions, delivery, diffusion, uptake and accumulation were investigated in this tumor cell model. Transmission electron microscopy (TEM) was utilized to characterize spheroids at various culture ages prior to the introduction of AuNPs. Imaging analysis was used to observe structural changes in response to treating spheroids with 45 nm AuNPs. Considerable diversity was observed in the size of the outermost of the spheroid. The nuclei of these cells were large, irregular nuclei with distributed heterochromatin and expanded nucleoli. The innermost cells had evidence of degeneration and necrosis, including organelle enlargement, cytoplasmic vacuolation, and large, digested debris vacuoles. Signs of necrosis were observed on Day 10 and became more evident on Day 20, especially in spheroids produced from 1000 cancer cells during culture. The cells had dispersed chromatin, large irregular nucleoli and nuclei, autophagic vacuoles, lysosomes close to the nucleus, and karyolysis of the nucleus and degeneration of the cytoplasmic organelles. AuNP-treated spheroids cells demonstrated greater uptake in the outermost, peripheral cells. Spheroids exposed to AuNPs exhibited cell shrinkage, cell membrane disruption, widened intercellular spaces, and nuclear fragmentation with nuclear membrane alterations.

The gold standard of microfluidic fabrication techniques, SU-8 patterning, requires photolithography equipment and facilities and is not suitable for 3D microfluidics. A 3D printer is more convenient and may achieve high resolutions comparable to conventional photolithography, but only with select materials. Alternatively, 5-axis computer numerical control (CNC) micro-milling machines can efficiently prototype structures with high resolutions, high aspect ratios, and non-planar geometries from a variety of materials. These machines, however, have not been catered for laboratory-based, small-batch microfluidics development and are largely inaccessible to researchers. In this paper, we present a new 5-axis CNC micro-milling machine specifically designed for prototyping 3D microfluidic channels, made affordable for research and laboratories. The machine is assembled from commercially available products and custom-build parts, occupying 0.72 cubic meters, and operating entirely from computer aided design (CAD) and manufacturing (CAM) software. The 5-axis CNC micro-milling machine achieves sub-mu m bidirectional repeatability (<= 0.23 mu m), machinable features <20 mu m, and a work volume of 50 x 50 x 68 mm. The tool compatibility and milling parameters were designed to enable fabrication of virtually any mill-able material including metals like aluminum, brass, stainless steel, and titanium alloys. To demonstrate milling high resolution and high aspect ratios, we milled a thin wall from 360 brass with a width of 18.1 mu m and an aspect ratio of similar to 50 : 1. We also demonstrated fabricating molds from 360 brass with non-planar geometries to create polydimethylsiloxane (PDMS) microfluidic channels. These included a channel on a 90 degrees edge and a channel on a rounded edge with a 250 mu m radius of curvature. Our 5-axis CNC micro-milling machine offers the most versatility in prototyping microfluidics by enabling high resolutions, geometric complexity, a large work volume, and broad material compatibility, all within a user-friendly benchtop system.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

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.

We have developed a novel microscopic analysis system that combines the functions of light-sheet fluorescence microscopy (LSM) and dynamic mechanical analysis (DMA). We have integrated the three uniquely designed components of (i) a MEMS dynamic compression device with a mu-force sensor, (ii) a high-speed 3D light-sheet scanner and an imager, and (iii) a customprogrammed image-based 3D modeling algorithm. Here, we demonstrate spatially-resolved mechanical characterization of viscoelastic materials under high-resolution 3D fluorescence microscopy for the first time.

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.

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.

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.

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.

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.

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.

Imaging of biological cells and tissues often relies on fluorescent labels, which offer high contrast with molecular specificity. The use of exogenous labeling agents, however, may alter the normal physiology of the bio-specimens. Complementary to the established fluorescence microscopy, label-free quantitative phase imaging provides an objective morphological measurement tool for bio-specimens and is free of variability introduced by contrast agents. Here we report a simple and low-cost microscope add-on, termed Ptychographic Modulation Engine (PME), for super-resolution quantitative phase imaging. In this microscope add-on module, we attach a diffuser to a 3D-printed holder that can be mechanically moved to different x-y positions. We then use two vibrational motors to introduce random positional shifts to the diffuser. The add-on module can be placed between the objective lens and the specimen in most existing microscope platforms. Thanks to the diffuser modulation process, the otherwise inaccessible high-resolution object information can now be encoded into the captured images. In the ptychographic phase retrieval process, we jointly recover the complex object wavefront, the complex diffuser profile, and the unknown positional shifts of the diffuser. We demonstrate a 4-fold resolution gain over the diffraction limit of the employed 2X objective lens. We also test our approach for in-vivo cell imaging, where we are able to adjust the focus after the data has been captured. The reported microscope add-on provides a turnkey solution for super-resolution quantitative phase imaging. It may find applications in label-free bio-imaging where both large field-of-view and high resolution are needed.

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.

Hydrocephalus is a disease in which a buildup of cerebral spinal fluid in the ventricles of the brain can cause brain trauma and death if untreated. The current standard of treatment is to insert a shunt to drain the fluid from the ventricles in the brain to the abdomen. A shortcoming of this approach is that the shunt may get clogged, and this failure is not easily detected. We have developed a novel microfluidic sensor for application in a cerebrospinal fluid shunt. The sensor system is totally passive and no implanted electronics are required. The sensor itself is a bending based cantilever made from biocompatible polydimethylsiloxane (PDMS) and is capable of measuring flow rates from 20 ml/hr to 120 ml/hr, which is the indicated range for the flow rate of cerebral spinal fluid. The sensor is paired with an optical detection system that uses a small light spot to read the changes in sensor position due to flow. The light is input from outside the brain into the implanted optical system and the output light is measured by a camera external to the body. The sensor stability was verified by running cerebral spinal fluid over the cantilever continuously for two weeks. The ability of the sensor to measure pulsed flow and linearly increasing flow rates was also verified. All of the materials used for the device are biocompatible materials and are amenable for manufacturing and animal tests and clinical studies. (C) 2020 Elsevier B.V. All rights reserved.