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The nematode worm Caenorhabditis elegans is a model organism widely used in biological research on genetics, development, neuroscience, and aging. Microinjection is an effective and widely adopted method to create transgenetic worms, perform ribonucleic acid (RNA) interference of certain genes, and introduce different types of molecules into specific locations inside a worm body. Based on microfluidics and robotic micromanipulation techniques, we develop a robotic system for automated microinjection of C. elegans with greatly improved injection speed and success rate over traditional manual microinjection. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for automated on-chip loading, immobilization, injection, and downstream sorting of single worms. A new autofocusing-based contact detection algorithm is proposed to find the optimal injection position along the depth direction of the microscope field of view. The direction and location of the needle tip are reliably identified using an image processing algorithm. Through experiments on 240 worms, the system demonstrates automated injection at a speed of 6 worms/min (9.97 s/worm) with a presorting operation success rate of 78.8% (postsorting operation success rate: 100%), which are more than 23 times faster and 1.6 times higher than the speed (0.25 worm/min) and success rate (30%) of a proficient human operator, respectively. With the superior performance, this system will enable new large-scale gene- and molecule-screening studies on C. elegans that cannot be fulfilled by the conventional microinjection technique. Note to Practitioners —In the worm biology community, there are thousands of research laboratories worldwide that routinely cope with worm microinjection experiments. This article aims to present the functionality and performance of our automated robotic system for high-speed worm injection. Using the robotic system, a large number of C. elegans can be loaded into the microfluidic device for continuous worm immobilization and injection. A user-friendly graphical user interface (GUI) is developed to allow an operator to monitor the injection process on a computer screen, select the injection location inside the worm body (through computer mouse clicking), and direct the system (through keyboard input) for downstream sorting of the successfully injected worms for further culture. Given its unique features, such as high injection speed, high level of automation, and high success/survival rates, this system holds great potential to liberate worm researchers from the tedious manual injection process and provide unparalleled injection throughput and consistency.

Paper has been a popular material of choice for biomedical applications including for bioanalysis and cell biology studies. Regular cellulose paper-based devices, however, have several key limitations including slow fluid flow; large sample retention in the paper matrix for microfluidic paper-based analytical device (μPAD) application; serious solvent evaporation issues, and contamination and poor control of experimental conditions for cell culture. Here, we describe the development of two novel platforms, nanopaper-based analytical devices (nanoPADs) and nanofibrillated adherent cell-culture platforms (nanoFACEs), that use nanofibrillated cellulose (NFC) paper, simply called nanopaper, as the substrate material to create transparent, pump-free and hollow-channel paper-based microfluidic devices. Due to the natural hydrophilicity and nanoscale pore size of nanopaper, the hollow-channel microfluidic devices can realize a totally pump-free flow without any complicated surface chemical functionalization on the nanopaper. Experimental results showed that within a certain range, larger hollow channel size leads to faster pump-free flows. Different from previous designs of paper-based hollow-channel microfluidic devices, the high transparency of the nanopaper substrate enabled the integration of various optical sensing and imaging technologies together with the nanoPADs and nanoFACEs. As proof-of-concept demonstrations, we demonstrated the use of nanoPADs for colorimetric sensing of glucose and surface-enhanced Raman spectroscopy (SERS)-based detection of environmental pollutants and applied the nanoFACEs to the culture of human umbilical vein endothelial cells (HUVECs). These demonstrations show the great promise of nanoPADs and nanoFACEs for biomedical applications such as chemical/bioanalysis and cell biology studies.

This paper reports a vision-based automated microfluidic system for morphological measurement and size-based sorting of the nematode worm C. elegans . Exceeding the capabilities of conventional worm sorting microfluidic devices purely relying on passive sorting mechanisms, our system is capable of accurate measurement of the worm length/width and active sorting of worms with the desired sizes from a mixture of worms with different body sizes. This function is realized based on the combination of real-time, vision-based worm detection and sizing algorithms and automated on-chip worm manipulation. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for sequential loading, trapping, vision-based sizing, and sorting of single worms. To keep the system operation robust, vision-based algorithms on detecting multi-worm loading and worm sizing failure have also been developed. We conducted sorting experiments on 319 worms and achieved an average sorting speed of 10.4 worms per minute (5.8 s/worm) with an operation success rate of 90.3%. This system will facilitate the worm biology studies where body size measurement and size-based sorting of many worms are needed.

The Drosophila larva is an excellent model organism in biology for studying the mechanisms of sensory mechanotransduction and the neural basis of behavior. This paper reports an automated robotic micromanipulation system capable of force-controlled mechanical stimulation and locomotion behavior analysis of freely moving Drosophila larvae, which improves the force regulation accuracy and larva operation consistency over conventional manual manipulation. A three-dimensionally-printed, three-axis micro force sensor was developed and integrated into the robotic system to measure the contact force between the tip of an end-effector and the larva head, and an adaptive fuzzy proportional-integral-derivative (PID) controller was proposed for closed-loop control of the contact force. The three-axis force sensor was also employed to monitor lateral disturbances to the contact force caused by small larva head movements, and thus validate the effectiveness of larva stimulation. The robotic system performed automated larva stimulation and locomotion analysis at a speed of four larvae per minute, and was applied to quantify the correlation between the applied contact force direction/magnitude and the larva reorientation behavior. With its high accuracy and efficiency, this system will greatly facilitate large-scale studies of mechanosensory behaviors in Drosophila larva.

Learning from the locomotion of natural organisms is one of the most effective strategies for designing microrobots. However, the development of bioinspired microrobots is still challenging because of technical bottlenecks such as design and seamless integration of high-performance actuation mechanism and high-density energy source for untethered locomotion. Directly harnessing the activation energy and intelligence of living tissues in synthetic micromachines provides an alternative route to developing biohybrid microrobots. Here, we propose an approach to engineering the genetic and nervous systems of a nematode worm, Caenorhabditis elegans, and creating an untethered, highly controllable living soft microrobot (called "RoboWorm"). A living worm is engineered through optogenetic and biochemical methods to shut down the signal transmissions between its neuronal and muscular systems while its muscle cells still remain optically excitable. Through dynamic modeling and experimental verification of the worm crawling, we found that the phase difference between the worm body curvature and the muscular activation pattern generates the thrust force for crawling locomotion. By reproducing the phase difference via optogenetic excitation of the worm body muscles, we emulated the major worm crawling behaviors in a controllable manner. Furthermore, with real-time visual feedback of the worm crawling, we realized closed-loop regulation of the movement direction and destination of single worms. This technology may facilitate scientific studies on the biophysics and neural basis of crawling locomotion of C. elegans and other nematode species.

Micromanipulation and immobilization of single cells and small organisms (e.g., Caenorhabditis elegans and Drosophila larva) are important experimental techniques in biological and biomedical research. Because of the micrometer sizes and highly fragile structures of cells and small organisms, conventional manipulation and immobilization techniques are not accurate and/or efficient enough to meet certain demanding needs in cellular and organismal studies. To this end, different types of microfluidic device have been developed to improve the accuracy, efficiency, and consistency of cell/organism immobilization and manipulation, which enables high-throughput single cell/organism analysis. In this chapter, we will discuss three microfluidic devices we previously developed for immobilization and robotic micromanipulation of single cells and small organisms. In closing, a conclusion and outlook into future trends is also provided.

Learning from the locomotion of natural organisms is one of the most effective strategies for designing microrobots. However, the development of bioinspired microrobots is still challenging because of technical bottlenecks such as design and seamless integration of high-performance actuation mechanism and high-density energy source for untethered locomotion. Directly harnessing the activation energy and intelligence of living tissues in synthetic micromachines provides an alternative route to developing biohybrid microrobots. Here, we propose an approach to engineering the genetic and nervous systems of a nematode worm

This paper reports a vision-based microfluidic system for automated, high-speed sorting of the nematode worm C. elegans. Exceeding the capabilities of conventional worm sorting microfluidic devices purely relying on passive sorting mechanisms, our system is capable of accurate measurement of the worm body length/width and active sorting of worms with the desired sizes from a mixture of worms at different developmental stages. This feature is enabled by the combination of vision-based worm detection and sizing algorithms and automated on-chip worm manipulation. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for sequential loading, trapping, imaging, and sorting of single worms based on vision-based worm size measurement results. To keep the system operation robust, vision-based algorithms for detecting multi-worm loading and worm size measurement failure have also been developed. We conducted sorting experiments on 319 worms and achieve an average sorting speed of 10.4 worms per minute (5.8 s/worm) with an operation success rate of 90.3%. This system will facilitate worm biology studies where body size measurement and size-based sorting of many worms are needed.

In this article, we propose a robotic rapid prototyping technology for fabricating a new type of paper-based field-effect transistors (FETs). Unlike the existing paper-based electronics developed primarily based on the printing of conductive and semiconductive inks on paper substrates, this new prototyping technology integrates single rolled-up semiconductive microtubes into a device with robotically printed electrodes as the semiconductive channel, through robotic micromanipulation techniques. To improve the uniformity of the printed silver (Ag) ink electrodes, we designed a time-shift mechanism to compensate for the nonuniform ink dispersion at the beginning and ending phases of the robotic printing process. In addition, image processing and motion control algorithms were developed to enable automatic transfer of the prefabricated microtube from its hosting silicon wafer to the printed electrodes on a paper substrate. The effectiveness of the proposed technology was verified by fabricating the paper-based FETs with semiconductive zinc oxide (ZnO) microtubes. Preliminary tests on the mobility of the fabricated ZnO FET demonstrated improved performance over the conventional paper-based FETs with printed semiconductive channels.

The Drosophila larva is an excellent model organism in biology for studying the mechanisms of sensory mechanotrans-duction and the neural basis of behavior. This paper reports an automated robotic system capable of force-controlled mechanical stimulation and locomotion behavior analysis of freely moving Drosophila larvae, which improves the force regulation accuracy and larva operation consistency over conventional manual manipulation. A 3D-printed, three-axis force sensor was developed and integrated into the robotic system to measure the contact force between the tip of an end-effector and the larva head, and an adaptive fuzzy proportional-integral-derivative (PID) controller was proposed for closed-loop control of the contact force. The three-axis force sensor was also employed to monitor lateral disturbances to the contact force caused by small larva head movements, and thus to validate the effectiveness of larva stimulation. The robotic system performed automated larva stimulation and locomotion analysis at a speed of four larvae per minute, and was applied to quantify the correlation between the applied contact force direction/magnitude and the larva reorientation behavior. With its high accuracy and efficiency, this system will greatly facilitate large-scale studies of mechanosensory behaviors in Drosophila larva.

The fabrication techniques of paper-based electronics have been widely explored in the past two decades, contributing to the wide application of these promising devices in a variety of fields. In this paper, we report a new rapid prototyping technique for constructing paper-based field-effect transistor (FET) biosensors integrating rolled-up semiconductor microtubes. Leveraging robotic printing and micromanipulation techniques, silver ink electrodes are printed on paper and pre-synthesized microtubes are transferred onto a pair of printed electrodes to form a complete FET. Compared with existing fabrication techniques of paper-based electronics that purely rely on printing, the proposed technique is more versatile in that it can rapidly integrate high-performance semiconductor microtubes onto paper substrates. To improve the printing uniformity, a time-shift mechanism is proposed to compensate the ink under-and over-dispersion at the beginning and end of the printing process, repsectively. To realize automated microtube pick-up and transfer, image processing and motion control algorithms are developed to detect positions of the end-effector (a glass needle) and the microtube, and to control motions of the glass needle with minimized human intervention. We demonstrate the effectiveness of the technique by fabricating the designed paper-based devices using bimetallic microtubes and measure their current-voltage characteristics.

This paper focuses on H ∞ filtering problem for a class of discrete-time switched nonlinear system under both fast and slow switching property. Each nonlinear mode of the switched system is expressed by a set of linear systems in local regions via T–S fuzzy modeling. Based on mode-dependent and fuzzy-basis-dependent Lyapunov functions, the existence conditions for the desired full-order filters are derived such that the developed filtering error system is globally uniformly asymptotically stable with a given H ∞ performance index. In particular, the mode-dependent average dwell time switching scheme is proposed for slow switching to relax the restrictions of average dwell time. Finally, the validity and potential of the developed theoretical results are demonstrated by a numerical example.

Force sensing and control are of paramount importance in robotic micromanipulation. A contact force regulator capable of accurately applying mechanical stimuli to a live Drosophila larva could greatly facilitate mechanobiology research on Drosophila and may eventually lead to novel discoveries in mechanotransduction mechanisms of neuronal circuitries. In this paper, we present a novel contact force control scheme implemented in an automated Drosophila larvae micromanipulation system, featuring a switched fuzzy to proportional-differential (PD) controller and a noise-insensitive extended high gain observer (EHGO). The switched fuzzy-PD control law inherits the fast convergence of fuzzy control and overcomes its drawbacks such as large overshoot and steady-state oscillation. The noise-insensitive EHGO can reliably estimate system modeling errors and is robust to force measurement noises, which is advantageous over conventional high gain observers (sensitive to signal noises). Force control experiments show that, compared to a proportional-integral-differential (PID) controller, this new force control scheme significantly enhances the system dynamic performance in terms of rising time, overshoot, and oscillation. The developed robotic system and the force control scheme will be applied to mechanical stimulation and fluorescence imaging of Drosophila larvae for identifying new mechanotransduction mechanisms.

Microindentation is a useful experimental technique for characterizing mechanical properties of soft materials for research in biomechanics, biomaterials, tissue engineering. Despite its powerful capabilities, the access to microindentation techniques is hampered by the low performance-to-cost ratio of current commercial microindentation systems. This paper describes a new approach for constructing microindentation systems from readily available laboratory resources, and reports a force-controlled, cost-effective microindentation system capable of elastic and viscoelastic characterization of soft materials. A micro-electro-mechanical systems (MEMS) based piezoresistive force sensor and a motorized micromanipulator are employed to indent a sample and collect the force-deformation data for extraction of elastic and viscoelastic parameters. To overcome the shortcomings of previously reported customized systems, closed-loop position and force controllers are designed and implemented to accurately regulate the indentation depth and force. Tests on elastomeric and hydrogel materials prove the effectiveness of the system for elastic, relaxation, and creep tests, providing comparable measurement results with commercial microindentation systems at a much lower cost.

Force sensing and control are of paramount importance in robotic micromanipulation. A contact force regulator capable of accurately applying mechanical stimuli to a live Drosophila larva could greatly facilitate mechanobiology research on Drosophila and may eventually lead to novel discoveries in mechanotransduction of neuron circuitry. In this paper, we present a novel contact force scheme implemented in an automated Drosophila larvae micromanipulation system, featuring a switched fuzzy-PD controller and a noise-insensitive extended high gain observer. The switched fuzzy-PD controller inherits the fast convergence from fuzzy controller and overcomes the drawbacks (overshoot and oscillation) of a conventional fuzzy controller. The observer provides precise estimation to compensate for system modeling errors despite force measurement noise, which overcomes the disadvantage of traditional high gain observer. Force control experiments show that, compared to an conventional PID controller, this new controller-observer scheme has significantly enhanced dynamic performance in terms of rising time, overshoot, and oscillation. The developed robotic system and the force control scheme will be applied to mechanical stimulation and fluorescence imaging of Drosophila larvae for identifying new mechanotransduction mechanisms.

The tiny nematode worm Caenorhabditis elegans has long been a popular model organism for genetic, developmental, and biochemical studies in which worm microinjection plays a critical role. This paper presents an automated robotic system for high-speed injection of C. elegans with an efficiency more than 10 times faster than that of a proficient injection technician. To facilitate the injection process, a multilayer, hydraulically-controlled polydimethylsiloxane (PDMS) microfluidic device is developed to rapidly load, immobilize, flush, sort and collect individual worms. In addition, a newly proposed contact detection algorithm is adopted to find the optimal injection position along the z axis within the microscope view field. The direction and location of the needle tip are identified online based on an effective image processing algorithm. According to continuous injection of 40 worms, our system is able to perform worm injection at a speed of 6.6 worms per minute with a pre-sorting success rate of 77.5% (post sorting: 100%). The superior performance provided by the system will significantly facilitate large-scale transgenic studies and biomolecule screening on C. elegans.

The nematode worm Caenorhabditis elegans has been widely used as a model organism in biological studies because of its short and prolific life cycle, relatively simple body structure, significant genetic overlap with human, and facile/inexpensive cultivation. Microinjection, as an established and versatile tool for delivering liquid substances into cellular/organismal objects, plays an important role in C. elegans research. However, the conventional manual procedure of C. elegans microinjection is labor-intensive and time-consuming and thus hinders large-scale C. elegans studies involving microinjection of a large number of C. elegans on a daily basis. In this paper, we report a novel microfluidic device that enables, for the first time, fully automated, high-speed microinjection of C. elegans. The device is automatically regulated by on-chip pneumatic valves and allows rapid loading, immobilization, injection, and downstream sorting of single C. elegans. For demonstration, we performed microinjection experiments on 200 C. elegans worms and demonstrated an average injection speed of 6.6 worm/min (average worm handling time: 9.45 s/worm) and a success rate of 77.5% (post-sorting success rate: 100%), both much higher than the performance of manual operation (speed: 1 worm/4 min and success rate: 30%). We conducted typical viability tests on the injected C. elegans and confirmed that the automated injection system does not impose significant adverse effect on the physiological condition of the injected C. elegans. We believe that the developed microfluidic device holds great potential to become a useful tool for facilitating high-throughput, large-scale worm biology research.