Cost-Effective Full-Color 3D Dental Imaging Based on Close-Range Photogrammetry.

Dental imaging plays a crucial role in clinical dental practice. Conventional 2D dental imaging serves general-purpose tasks, such as patient documentation, while high-precision 3D dental scanning is tailored for specialized procedures, such as orthodontics and implant surgeries. In this study, we aimed to develop a cost-effective 3D imaging technique that could bridge the gap between conventional dental photography and high-precision 3D dental scanning, with the goal of improving patient dental care. We developed a 3D imaging technique based on close-range photogrammetry and termed it close-range photogrammetry-based dental imaging (CPDI). We evaluated this technique on both in vitro dental models and in vivo teeth. For dental models, we conducted a parametric study to examine the effects of the depth of field and specular reflection on reconstruction quality. We showed that the optimal results were achieved with an f/5.6 lens and without a circular polarizer for reflection suppression. This configuration generated 3D scans with 57.7 ± 3.2% and 82.4 ± 2.7% of reconstructed points falling within ±0.1 mm and ±0.2 mm error margins, respectively. With such accuracy, these 3D dental models can faithfully represent dental morphology and features. During in vivo imaging, we were able to reconstruct high-quality 3D models of the anterior arch, further demonstrating its clinical relevance. The reconstructed models carry both 3D shapes and detail full-color surface textures, which positions CPDI as a versatile imaging tool in different areas of clinical dental care.

Optimizing Sensitivity in a Fluid-Structure Interaction-Based Microfluidic Viscometer: A Multiphysics Simulation Study.

Fluid-structure interactions (FSI) are used in a variety of sensors based on micro- and nanotechnology to detect and measure changes in pressure, flow, and viscosity of fluids. These sensors typically consist of a flexible structure that deforms in response to the fluid flow and generates an electrical, optical, or mechanical signal that can be measured. FSI-based sensors have recently been utilized in applications such as biomedical devices, environmental monitoring, and aerospace engineering, where the accurate measurement of fluid properties is critical to ensure performance and safety. In this work, multiphysics models are employed to identify and study parameters that affect the performance of an FSI-based microfluidic viscometer that measures the viscosity of Newtonian and non-Newtonian fluids using the deflection of flexible micropillars. Specifically, we studied the impact of geometric parameters such as pillar diameter and height, aspect ratio of the pillars, pillar spacing, and the distance between the pillars and the channel walls. Our study provides design guidelines to adjust the sensitivity of the viscometer toward specific applications. Overall, this highly sensitive microfluidic sensor can be integrated into complex systems and provide real-time monitoring of fluid viscosity.

A micropillar-based microfluidic viscometer for Newtonian and non-Newtonian fluids.

In this study, a novel viscosity measurement technique based on measuring the deflection of flexible (poly) dimethylsiloxane (PDMS) micropillars is presented. The experimental results show a nonlinear relationship between fluid viscosity and the deflection of micropillars due to viscoelastic properties of PDMS. A calibration curve, demonstrating this nonlinear relationship, is generated, and used to determine the viscosity of an unknown fluid. Using our method, viscosity measurements for Newtonian fluids (glycerol/water solutions) can be performed within 2-100 cP at shear rates γ = 60.5-398.4 s-1. We also measured viscosity of human whole blood samples (non-Newtonian fluid) yielding 2.7-5.1 cP at shear rates γ = 120-345.1 s-1, which compares well with measurements using conventional rotational viscometers (3.6-5.7 cP). With a sensitivity better than 0.5 cP, this method has the potential to be used as a portable microfluidic viscometer for real-time rheological studies.

Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes.

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

The human gut is inhabited with hundreds of billions of bacterial cells from a wide range of families. This complex mixture of bacteria is part of the gut microbiome, along with other lifeforms such as viruses, archaea and fungi. As well as interacting with each other, the bacteria in the microbiome interact with our cells and available nutrients. Studying these interactions can help us understand how this community of bacteria influence health and disease. One way to study the diversity of the microbiome is to take a sample, such as a section of stool, and perform DNA sequencing to determine which types of bacteria are present. This can reveal how the composition of the gut microbiome relates to our health, but cannot confirm whether these bacteria are the cause or the effect of most diseases. To overcome this problem, researchers need to be able to grow pure strains of these bacteria in order to unravel their underlying mechanisms. For over a century, the conventional way to cultivate bacteria has been to grow them in a Petri dish. However, this method promotes the growth of more abundant, fast-growing bacterial strains. This results in a huge disconnect between the bacteria grown in a Petri dish and the diversity within the human gut, which is hindering our understanding of gut health and disease. Now, Watterson et al. have built a machine that improves the speed and number of cultivated bacterial organisms, thus paving the way for more detailed investigations of the human gut microbiome. This new system works by growing bacteria in millions of miniscule droplets which can be physically separated to help the expansion of slower growing species. Watterson et al. cultivated bacterial cells from a stool sample from a single donor using the droplet system and compared this to traditional culturing methods. The droplet technology increased the number of different organisms that were able to grow by up to four times, including those that were rare or slow-growing. Bacteria in the donor stool were then screened for populations that were resistant to antibiotics. This identified 21 antibiotic resistant bacteria which only grew in the droplets and not in Petri dishes. This droplet-based technology will make it possible to study bacterial strains that were previously difficult to grow. Furthermore, this method could help identify whether stool from a donor contains any antibiotic resistant strains, which can lead to clinical complications once transplanted. In future, this new technology could be used in laboratories or hospitals to study the role of the gut microbiome in health and disease.

Viable cell culture in PDMS-based microfluidic devices.

Microfluidics has played a vital role in developing novel methods to investigate biological phenomena at the molecular and cellular level during the last two decades. Microscale engineering of cellular systems is nevertheless a nascent field marked inherently by frequent disruptive advancements in technology such as PDMS-based soft lithography. Viable culture and manipulation of cells in microfluidic devices requires knowledge across multiple disciplines including molecular and cellular biology, chemistry, physics, and engineering. There has been numerous excellent reviews in the past 15 years on applications of microfluidics for molecular and cellular biology including microfluidic cell culture (Berthier et al., 2012; El-Ali, Sorger, & Jensen, 2006; Halldorsson et al., 2015; Kim et al., 2007; Mehling & Tay, 2014; Sackmann et al., 2014; Whitesides, 2006; Young & Beebe, 2010), cell culture models (Gupta et al., 2016; Inamdar & Borenstein, 2011; Meyvantsson & Beebe, 2008), cell secretion (Schrell et al., 2016), chemotaxis (Kim & Wu, 2012; Wu et al., 2013), neuron culture (Millet & Gillette, 2012a, 2012b), drug screening (Dittrich & Manz, 2006; Eribol, Uguz, & Ulgen, 2016; Wu, Huang, & Lee, 2010), cell sorting (Autebert et al., 2012; Bhagat et al., 2010; Gossett et al., 2010; Wyatt Shields Iv, Reyes, & López, 2015), single cell studies (Lecault et al., 2012; Reece et al., 2016; Yin & Marshall, 2012), stem cell biology (Burdick & Vunjak-Novakovic, 2009; Wu et al., 2011; Zhang & Austin, 2012), cell differentiation (Zhang et al., 2017a), systems biology (Breslauer, Lee, & Lee, 2006), 3D cell culture (Huh et al., 2011; Li et al., 2012; van Duinen et al., 2015), spheroids and organoids (Lee et al., 2016; Montanez-Sauri, Beebe, & Sung, 2015; Morimoto & Takeuchi, 2013; Skardal et al., 2016; Young, 2013), organ-on-chip (Bhatia & Ingber, 2014; Esch, Bahinski, & Huh, 2015; Huh et al., 2011; van der Meer & van den Berg, 2012), and tissue engineering (Andersson & Van Den Berg, 2004; Choi et al., 2007; Hasan et al., 2014). In this chapter, we provide an overview of PDMS-based microdevices for microfluidic cell culture. We discuss the advantages and challenges of using PDMS-based soft lithography for microfluidic cell culture and highlight recent progress and future directions in this area.

Enhanced Dissolution of Liquid Microdroplets in the Extensional Creeping Flow of a Hydrodynamic Trap.

A novel noncontact technique based on hydrodynamic trapping is presented to study the dissolution of freely suspended liquid microdroplets into a second immiscible phase in a simple extensional creeping flow. Benzyl benzoate (BB) and n-decanol microdroplets are individually trapped at the stagnation point of a planar extensional flow, and dissolution of single microdroplets into an aqueous solution containing surfactant is characterized at different flow rates. The experimental dissolution curves are compared to two models: (i) the Epstein-Plesset (EP) model which considers only diffusive mass transfer, and (ii) the Zhang-Yang-Mao (ZYM) model which considers both diffusive and convective mass transfer in the presence of extensional creeping flow. The EP model significantly underpredicts the experimentally determined dissolution rates for all experiments. In contrast, very good agreement is observed between the experimental dissolution curves and the ZYM model when the saturation concentration of the microdroplet liquid (cs) is used as the only fitting parameter. Experiments with BB microdroplets at low surfactant concentration (10 µM) reveal cs values very similar to that reported in the literature. In contrast, experiments with BB and n-decanol microdroplets at 10 mM surfactant concentration, higher than the critical micelle concentration (CMC) of 5 mM, show further enhancements in microdroplet dissolution rates due to micellar solubilization. The presented method accurately tests the dissolution of single microdroplets into a second immiscible phase in extensional creeping flow and has potential for applications such as separation processes, food dispersion, and drug development/design.

Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution.

We report an automated microfluidic-based platform for single cell analysis that allows for cell culture in free solution with the ability to control the cell growth environment. Using this approach, cells are confined by the sole action of gentle fluid flow, thereby enabling non-perturbative analysis of cell growth away from solid boundaries. In addition, the single cell microbioreactor allows for precise and time-dependent control over cell culture media, with the combined ability to observe the dynamics of non-adherent cells over long time scales. As a proof-of-principle demonstration, we used the platform to observe dynamic cell growth, gene expression, and intracellular diffusion of repressor proteins while precisely tuning the cell growth environment. Overall, this microfluidic approach enables the direct observation of cellular dynamics with exquisite control over environmental conditions, which will be useful for quantifying the behaviour of single cells in well-defined media.

Fluidic-directed assembly of aligned oligopeptides with π-conjugated cores.

A microfluidic-based directed assembly strategy is employed to form highly aligned supramolecular structures. Formation of aligned synthetic oligopeptide nanostructures is accomplished using planar extensional flow, which induces alignment of underlying material suprastructures. Fluidic-directed assembly of supramolecular structures allows for unprecedented manipulation at the nano- and mesoscales, which has the potential to provide rapid and efficient control of functional material properties.

Manipulation and confinement of single particles using fluid flow.

High precision control of micro- and nanoscale objects in aqueous media is an essential technology for nanoscience and engineering. Existing methods for particle trapping primarily depend on optical, magnetic, electrokinetic, and acoustic fields. In this work, we report a new hydrodynamic flow based approach that allows for fine-scale manipulation and positioning of single micro- and nanoscale particles using automated fluid flow. As a proof-of-concept, we demonstrate trapping and two-dimensional (2D) manipulation of 500 nm and 2.2 µm diameter particles with a positioning precision as small as 180 nm during confinement. By adjusting a single flow parameter, we further show that the shape of the effective trap potential can be efficiently controlled. Finally, we demonstrate two distinct features of the flow-based trapping method, including isolation of a single particle from a crowded particle solution and active control over the surrounding medium of a trapped object. The 2D flow-based trapping method described here further expands the micro/nanomanipulation toolbox for small particles and holds strong promise for applications in biology, chemistry, and materials research.

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability.

Microfluidic Wheatstone bridge for rapid sample analysis.

We developed a microfluidic analogue of the classic Wheatstone bridge circuit for automated, real-time sampling of solutions in a flow-through device format. We demonstrate precise control of flow rate and flow direction in the "bridge" microchannel using an on-chip membrane valve, which functions as an integrated "variable resistor". We implement an automated feedback control mechanism in order to dynamically adjust valve opening, thereby manipulating the pressure drop across the bridge and precisely controlling fluid flow in the bridge channel. At a critical valve opening, the flow in the bridge channel can be completely stopped by balancing the flow resistances in the Wheatstone bridge device, which facilitates rapid, on-demand fluid sampling in the bridge channel. In this article, we present the underlying mechanism for device operation and report key design parameters that determine device performance. Overall, the microfluidic Wheatstone bridge represents a new and versatile method for on-chip flow control and sample manipulation.

A microfluidic-based hydrodynamic trap: design and implementation.

We report an integrated microfluidic device for fine-scale manipulation and confinement of micro- and nanoscale particles in free-solution. Using this device, single particles are trapped in a stagnation point flow at the junction of two intersecting microchannels. The hydrodynamic trap is based on active flow control at a fluid stagnation point using an integrated on-chip valve in a monolithic PDMS-based microfluidic device. In this work, we characterize device design parameters enabling precise control of stagnation point position for efficient trap performance. The microfluidic-based hydrodynamic trap facilitates particle trapping using the sole action of fluid flow and provides a viable alternative to existing confinement and manipulation techniques based on electric, optical, magnetic or acoustic force fields. Overall, the hydrodynamic trap enables non-contact confinement of fluorescent and non-fluorescent particles for extended times and provides a new platform for fundamental studies in biology, biotechnology and materials science.

Multiplexed detection of nucleic acids in a combinatorial screening chip.

Multiplexed diagnostic testing has the potential to dramatically improve the quality of healthcare. Simultaneous measurement of health indicators and/or disease markers reduces turnaround time and analysis cost and speeds up the decision making process for diagnosis and treatment. At present, however, most diagnostic tests only provide information on a single indicator or marker. Development of efficient diagnostic tests capable of parallel screening of infectious disease markers could significantly advance clinical and diagnostic testing in both developed and developing parts of the world. Here, we report the multiplexed detection of nucleic acids as disease markers within discrete wells of a microfluidic chip using molecular beacons and total internal reflection fluorescence microscopy (TIRFM). Using a 4 × 4 array of 200 pL wells, we screened for the presence of four target single stranded oligonucleotides encoding for conserved regions of the genomes of four common viruses: human immunodeficiency virus-1 (HIV-1), human papillomavirus (HPV), Hepatitis A (Hep A) and Hepatitis B (Hep B). Target oligonucleotides are accurately detected and discriminated against alternative oligonucleotides with different sequences. This combinatorial chip represents a versatile platform for the development of clinical diagnostic tests for simultaneous screening, detection and monitoring of a wide range of biological markers of disease and health using minimal sample size.

A microfluidic-based hydrodynamic trap for single particles.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

Hydrodynamic trap for single particles and cells.

Trapping and manipulation of microscale and nanoscale particles is demonstrated using the sole action of hydrodynamic forces. We developed an automated particle trap based on a stagnation point flow generated in a microfluidic device. The hydrodynamic trap enables confinement and manipulation of single particles in low viscosity (1-10 cP) aqueous solution. Using this method, we trapped microscale and nanoscale particles (100 nm-15 mum) for long time scales (minutes to hours). We demonstrate particle confinement to within 1 mum of the trap center, corresponding to a trap stiffness of approximately 10(-5)-10(-4) pNnm.

Detecting Single Bacterial Cells through Optical Resonances in Microdroplets.

Detection of single bacterial cells through optical resonances has been demonstrated in microdroplets. The setup enables high throughput non-specific detection of single E. Coli cells without any labeling. The cells inside the microdroplet have a direct effect on the morphology dependent resonances that are supported by Rhodamine 6G fluorescence; modification of the resonances arises from changes due to scattering and changes in the local refractive index. The change in the optical resonance spectrum can be observed at low concentrations where each microdroplet contains no more than one cell.

Lasing droplets in a microfabricated channel.

Lasing from spherical microdroplets ejected into a liquid medium with a lower refractive index is observed in a microchannel. A microfabricated device that combines droplet production and excitation/detection has been utilized. Droplets of 50 microm diameter containing a fluorescent dye were first detected and then excited through multimode fibers after their production at a T-junction. Images show intense lasing emission around the droplet rim. Spectra from the droplets exhibit morphology-dependent resonances that are redshifted relative to the bulk fluorescence emission from the dyes. The dependence of resonant peak intensities on the pump beam power is nonlinear.