Orthogonal-view Microscope for the Biomechanics Investigations of Aquatic Organisms

Microscopes are essential for biomechanics and hydrodynamical investigation of small aquatic organisms. We report a DIY microscope (GLUBscope) that enables the visualization of organisms from two orthogonal imaging planes (top and side views). Compared to conventional imaging systems, this approach provides a comprehensive visualization strategy of organisms, which could have complex shapes and morphologies. The microscope was constructed by combining custom 3D-printed parts and off-the-shelf components. The system is designed for modularity and reconfigurability. Open-source design files and build instructions are provided in this report. Additionally, proof of use experiments, particularly with Hydra and other organisms that combine the GLUBscope with an analysis pipeline, were demonstrated. Beyond the applications demonstrated, the system can be used or modified for various imaging applications

Microfluidics for Hydrodynamics Investigations of Sand Dollar Larvae

The life cycle of most marine invertebrates includes a planktonic larval stage before metamorphosis to bottom-dwelling adulthood. During larval stage, ciliary-mediated activity enables feeding (capture unicellular algae) and transport of materials (oxygen) required for the larva's growth, development, and successful metamorphosis. Investigating the underlying hydrodynamics of these behaviors is valuable for addressing fundamental biological questions (e.g., phenotypic plasticity) and advancing engineering applications. In this work, we combined microfluidics and fluorescence microscopy as a miniaturized PIV (mPIV) to study ciliary-medicated hydrodynamics during suspension feeding in sand dollar larvae (Dendraster excentricus). First, we confirmed the approach's feasibility by examining the underlying hydrodynamics (vortex patterns) for low- and high-fed larvae. Next, ciliary hydrodynamics were tracked from 11 days post-fertilization (DPF) to 20 DPF for 21 low-fed larvae. Microfluidics enabled the examination of baseline activities (without external flow) and behaviors in the presence of environmental cues (external flow). A library of qualitative vortex patterns and quantitative hydrodynamics was generated and shared as a stand alone repository. Results from mPIV (velocities) were used to examine the role of ciliary activity in transporting materials (oxygen). Given the laminar flow and the viscosity-dominated environments surrounding the larvae, overcoming the diffusive boundary layer is critical for the organism's survival. Peclet number analysis for oxygen transport suggested that ciliary velocities help overcome the diffusion dominated transport (max Pe numbers between 30-60). Microfluidics serving as mPIV provided a scalable and accessible approach for investigating the ciliary hydrodynamics of marine organisms.Comment: 21 pages and 11 figures (videos not included

Microfluidics Generation of Millimeter-sized Matrigel Droplets

Significant progress has been made to increase access to droplet microfluidics for labs with limited microfluidics expertise or fabrication equipment. In particular, using off-the-shelf systems has been a valuable approach. However, the ability to modify a channel design and, thus, the functional characteristics of the system is of great value. In this work, we describe the development of co-flow microfluidics and their fabrication methods for generating uniform millimeter-sized (0.5 - 2 mm) hydrogel droplets. Two complementary approaches based on desktop CO2 laser cutting were developed to prototype and build durable co-flow droplet microfluidics. After demonstrating the co-flow systems, water-in-oil experiments and dimensionless number analysis were used to examine the operational characteristics of the system. Specifically, the Capillary number analysis indicated that millimeter-sized droplet generators operated in the desirable geometry-controlled regime despite their length scales being larger than traditional microfluidics systems. Next, the tunable generation of Matrigel droplets was demonstrated. By adjusting the relative flow rates, the droplet size could be tuned. Finally, we demonstrated fibroblast encapsulation and cell viability for up to 7 days as a proof-of-concept experiment. The systems presented are simple and effective tools to generate robust hydrogel droplets and increase the accessibility of this technology to teaching labs or research settings with limited resources or access to microfluidics.Comment: 14 pages, 5 figures, 1 tabl

A Programmable Microfluidic Finite State Machine for the Autonomous Lab on a Chip

Microfluidics is an adaptation of semiconductor technology to the creation of circuits of gas and liquid. They have enabled the establishment of mechanical and liquid circuits with complexity similar to integrated electrical circuits. However, while the microfluidic chips have been miniaturized, their external governing systems have remained unchanged. This lack of embedded control transforms the small chip into a large and often cumbersome system. Next generation of microfluidic systems will allow reduction or removal of these controllers. Various successful microfluidics for reduction of these external requirements has been demonstrated. However, an autonomous, self-contained, programmable microfluidic finite state machines (FSM) that only requires power to operate has remained absent.In this work, we present sequential logic circuits implemented in microfluidics rather than electronics for the autonomous control of liquid networks. We demonstrate microcontrollers, simple pneumatic computers, built entirely out of microfluidic parts. We also demonstrate a programmable FSM, first programmable microfluidic computer, built out of pneumatic Boolean logic gates and channels. We show a 6-bit asynchronous pneumatic counters, useful as an embedded timing reference. Added to the controllers we create liquid systems such as a 7 stage 1:1 serial diluter system, i.e. serial dilution ladder. Finally, we integrate liquid networks with these controllers to create self-contained microfluidic systems

A Programmable Microfluidic Finite State Machine for the Autonomous Lab on a Chip

Microfluidics is an adaptation of semiconductor technology to the creation of circuits of gas and liquid. They have enabled the establishment of mechanical and liquid circuits with complexity similar to integrated electrical circuits. However, while the microfluidic chips have been miniaturized, their external governing systems have remained unchanged. This lack of embedded control transforms the small chip into a large and often cumbersome system. Next generation of microfluidic systems will allow reduction or removal of these controllers. Various successful microfluidics for reduction of these external requirements has been demonstrated. However, an autonomous, self-contained, programmable microfluidic finite state machines (FSM) that only requires power to operate has remained absent.In this work, we present sequential logic circuits implemented in microfluidics rather than electronics for the autonomous control of liquid networks. We demonstrate microcontrollers, simple pneumatic computers, built entirely out of microfluidic parts. We also demonstrate a programmable FSM, first programmable microfluidic computer, built out of pneumatic Boolean logic gates and channels. We show a 6-bit asynchronous pneumatic counters, useful as an embedded timing reference. Added to the controllers we create liquid systems such as a 7 stage 1:1 serial diluter system, i.e. serial dilution ladder. Finally, we integrate liquid networks with these controllers to create self-contained microfluidic systems

Pneumatic computers for embedded control of microfluidics.

Alternative computing approaches that interface readily with physical systems are well suited for embedded control of those systems. We demonstrate finite state machines implemented as pneumatic circuits of microfluidic valves and use these controllers to direct microfluidic liquid handling procedures on the same chip. These monolithic integrated systems require only power to be supplied externally, in the form of a vacuum source. User input can be provided directly to the chip by covering pneumatic ports with a finger. State machines with up to four bits of state memory are demonstrated, and next-state combinational logic can be fully reprogrammed by changing the hole-punch pattern on a membrane in the chip. These pneumatic computers demonstrate a framework for the embedded control of physical systems and open a path to stand-alone lab-on-a-chip devices capable of highly complex functionality

sideSPIM – selective plane illumination based on a conventional inverted microscope

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system

Nanopillared Surfaces Disrupt Pseudomonas aeruginosa Mechanoresponsive Upstream Motility.

Pseudomonas aeruginosa is an opportunistic, multidrug-resistant, human pathogen that forms biofilms in environments with fluid flow, such as the lungs of cystic fibrosis patients, industrial pipelines, and medical devices. P. aeruginosa twitches upstream on surfaces by the cyclic extension and retraction of its mechanoresponsive type IV pili motility appendages. The prevention of upstream motility, host invasion, and infectious biofilm formation in fluid flow systems remains an unmet challenge. Here, we describe the design and application of scalable nanopillared surface structures fabricated using nanoimprint lithography that reduce upstream motility and colonization by P. aeruginosa. We used flow channels to induce shear stress typically found in catheter tubes and microscopy analysis to investigate the impact of nanopillared surfaces with different packing fractions on upstream motility trajectory, displacement, velocity, and surface attachment. We found that densely packed, subcellular nanopillared surfaces, with pillar periodicities ranging from 200 to 600 nm and widths ranging from 70 to 215 nm, inhibit the mechanoresponsive upstream motility and surface attachment. This bacteria-nanostructured surface interface effect allows us to tailor surfaces with specific nanopillared geometries for disrupting cell motility and attachment in fluid flow systems