5-Axis CNC micro-milling machine for three-dimensional microfluidics.

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.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during ovulation.

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 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.

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.

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.

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.

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.

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.

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.

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.

Design, Fabrication, and Validation of a Petri Dish-Compatible PDMS Bioreactor for the Tensile Stimulation and Characterization of Microtissues.

In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials.

Autofocusing technologies for whole slide imaging and automated microscopy.

Whole slide imaging (WSI) has moved digital pathology closer to diagnostic practice in recent years. Due to the inherent tissue topography variability, accurate autofocusing remains a critical challenge for WSI and automated microscopy systems. The traditional focus map surveying method is limited in its ability to acquire a high degree of focus points while still maintaining high throughput.

Studies of nanoparticle delivery with 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, testing with a model delivery system is crucial because it allows for real-time, quantitative transport analysis, which is often difficult with 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.

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.

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.

The Actuation Mechanism of 3D Printed Flexure-Based Robotic Microtweezers.

We report on the design and the modeling of a three-dimensional (3D) printed flexure-based actuation mechanism for robotic microtweezers, the main body of which is a single piece of nylon. Our design aims to fill a void in sample manipulation between two classes of widely used instruments: nano-scale and macro-scale robotic manipulators. The key component is a uniquely designed cam flexure system, which linearly translates the bending of a piezoelectric bimorph actuator into angular displacement.

Elastography of multicellular spheroids using 3D light microscopy.

We have demonstrated a new method of 3D elastography based on 3D light microscopy and micro-scale manipulation. We used custom-built micromanipulators to apply a mechanical force onto multicellular tumor spheroids (200-300 µm in size) and recorded the induced compression with a differential interference contrast (DIC)/confocal microscope to obtain a 4D (x, y, z, and indentation steps) image sequence. Deformation analysis made through 3D pattern tracking without using fluorescence revealed 3D structural and spatial heterogeneity in tumor spheroids.

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.

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.

Focused Ion Beam-Based Milling, Imaging and Analysis of 3D Tumor Spheroids.

We investigate the structural cellular alterations in breast cancer spheroids at various growth stages using transmission electron microscopy (TEM), focused ion beam (FIB), and scanning electron microscopy (SEM) imaging. Samples sliced by FIB milling were studied for 3D analysis and construction. The imaging results of different spheroid ages were compared for a better understanding of cancer spheroid models.

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

. 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.

Nondestructive, Label-Free Characterization of Mechanical Microheterogeneity in Biomimetic Materials.

We propose a novel nondestructive, label-free, mechanical characterization method for composite biomimetic materials. The method combines microscale-force measurement, bright-field microscopy based deformation analysis, and finite-element methods (FEM) to study the heterogeneity in bioengineered composite materials. The method was used to study silk fibroin protein based, donut-shaped scaffolds consisting of a shell (diameter 5 mm) and a core (diameter 2 mm) with a stiff-core or a soft-core configuration.

Stiffness analysis of 3D spheroids using microtweezers.

We describe a novel mechanical characterization method that has directly measured the stiffness of cancer spheroids for the first time to our knowledge. Stiffness is known to be a key parameter that characterizes cancerous and normal cells. Atomic force microscopy or optical tweezers have been typically used for characterization of single cells with the measurable forces ranging from sub pN to a few hundred nN, which are not suitable for measurement of larger 3D cellular structures such as spheroids, whose mechanical characteristics have not been fully studied.

Bioactive polymeric scaffolds for tissue engineering.

A variety of engineered scaffolds have been created for tissue engineering using polymers, ceramics and their composites. Biomimicry has been adopted for majority of the three-dimensional (3D) scaffold design both in terms of physicochemical properties, as well as bioactivity for superior tissue regeneration. Scaffolds fabricated via salt leaching, particle sintering, hydrogels and lithography have been successful in promoting cell growth and tissue regeneration .

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.

Single-frame rapid autofocusing for brightfield and fluorescence whole slide imaging.

A critical consideration for whole slide imaging (WSI) platform is to perform accurate autofocusing at high speed. Typical WSI systems acquire a z-stack of sample images and determine the best focal position by maximizing a figure of merit. This strategy, however, has suffered from several limitations, including low speed due to multiple image acquisitions, relatively low accuracy of focal plane estimation, short axial range for autofocusing, and difficulties in handling transparent samples.