Hybrid Microfluidic Devices For On-Demand Manipulation and Screening of Neurons and Organs of Small Model Organisms

Caenorhabditis elegans and Drosophila melanogaster are widely used model organisms for neurological and cardiac studies due to their simple neuronal and cardiac systems, genome similarity to humans, and ease of maintenance in laboratories. However, their 50m-1mm sizes and continuous mobility impede their precise spatiotemporal manipulation, thereby, reducing the throughput of biological assays. By integrating glass capillaries into microfluidic devices and using 3D-printed fixtures for precise control, we have developed hybrid lab-on-a-chip devices to facilitate the processes of animal manipulation and stimuli control, using modules for single-organism selection, orientation, imaging and chemical stimulation. These microdevices enabled us to manipulate organisms individually and to orient them at any desired direction for imaging purposes. The applications of these hybrid microdevices were demonstrated in the optical and fluorescent imaging of C. elegans cells as well as cardiac screening of Drosophila larvae. This technique can be applied in fundamental biology, toxicology, and drug discovery

Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alg

Hybrid optical and magnetic manipulation of microrobots

Microrobotic systems have the potential to provide precise manipulation on cellular level for diagnostics, drug delivery and surgical interventions. These systems vary from tethered to untethered microrobots with sizes below a micrometer to a few microns. However, their main disadvantage is that they do not have the same capabilities in terms of degrees-of-freedom, sensing and control as macroscale robotic systems. In particular, their lack of on-board sensing for pose or force feedback, their control methods and interface for automated or manual user control are limited as well as their geometry has few degrees-of-freedom making three-dimensional manipulation more challenging. This PhD project is on the development of a micromanipulation framework that can be used for single cell analysis using the Optical Tweezers as well as a combination of optical trapping and magnetic actuation for recon gurable microassembly. The focus is on untethered microrobots with sizes up to a few tens of microns that can be used in enclosed environments for ex vivo and in vitro medical applications. The work presented investigates the following aspects of microrobots for single cell analysis: i) The microfabrication procedure and design considerations that are taken into account in order to fabricate components for three-dimensional micromanipulation and microassembly, ii) vision-based methods to provide 6-degree-offreedom position and orientation feedback which is essential for closed-loop control, iii) manual and shared control manipulation methodologies that take into account the user input for multiple microrobot or three-dimensional microstructure manipulation and iv) a methodology for recon gurable microassembly combining the Optical Tweezers with magnetic actuation into a hybrid method of actuation for microassembly.Open Acces

A centrifugal microfluidic platform for capturing, assaying and manipulation of beads and biological cells

Microfluidics is deemed a field with great opportunities, especially for applications in medical diagnostics. The vision is to miniaturize processes typically performed in a central clinical lab into small, simple to use devices - so called lab-on-a-chip (LOC) systems. A wide variety of concepts for liquid actuation have been developed, including pressure driven flow, electro-osmotic actuation or capillary driven methods. This work is based on the centrifugal platform (lab-on-a-disc). Fluid actuation is performed by the forces induced due to the rotation of the disc, thus eliminating the need for external pumps since only a spindle motor is necessary to rotate the disc and propel the liquids inside of the micro structures. Lab-on-a-disc systems are especially promising for point-of-care applications involving particles or cells due to the centrifugal force present in a rotating system. Capturing, assaying and identification of biological cells and microparticles are important operations for lab-on-a-disc platforms, and the focus of this work is to provide novel building blocks towards an integrated system for cell and particle based assays. As a main outcome of my work, a novel particle capturing and manipulation scheme on a centrifugal microfluidic platform has been developed. To capture particles (biological cells or micro-beads) I designed an array of V-shaped micro cups and characterized it. Particles sediment under stagnant flow conditions into the array where they are then mechanically trapped in spatially well-defined locations. Due to the absence of flow during the capturing process, i.e. particle sedimentation is driven by the artificial gravity field on the centrifugal platform, the capture efficiency of this approach is close to 100% which is notably higher than values reported for typical pressure driven systems. After capturing the particles, the surrounding medium can easily be exchanged to expose them to various conditions such as staining solutions or washing buffers, and thus perform assays on the captured particles. By scale matching the size of the capturing elements to the size of the particles, sharply peaked single occupancy can be achieved. Since all particles are arrayed in the same focal plane in spatially well defined locations, operations such as counting or fluorescent detection can be performed easily. The application of this platform to perform multiplexed bead-based immunoassays as well as the discrimination of various cell types based on intra cellular and membrane based markers using fluorescently tagged antibodies is demonstrated. Additionally, methods to manipulate captured particles either in batch mode or on an individual particle level have been developed and characterized. Batch release of captured particles is performed by a novel magnetic actuator which is solely controlled by the rotation frequency of the disc. Furthermore, the application of this actuator to rapidly mix liquids is shown. Manipulation of individual particles is performed using an optical tweezers setup which has been developed as part of this work. Additionally, this optical module also provides fluorescence detection capabilities. This is the first time that optical tweezers have been combined with a centrifugal microfluidic system. This work presents the core technology for an integrated centrifugal platform to perform cell and particle based assays for fundamental research as well as for point-of- care applications. The key outputs of my specific work are: 1. Design, fabrication and characterization of a novel particle capturing scheme on a centrifugal microfluidic platform (V-cups) with very high capture efficiency (close to 100%) and sharply peaked single occupancy (up to 99.7% single occupancy). 2. A novel rotation frequency controlled magnetic actuator for releasing captured particles as well as for rapidly mixing liquids has been developed, manufactured and characterized. 3. The V-cup platform has successfully been employed to capture cells and perform multi-step antibody staining assays for cell discrimination. 4. An optical tweezers setup has been built and integrated into a centrifugal teststand, and successful manipulation of individual particles trapped in the V-cup array is demonstrated

Roadmap for Optical Tweezers 2023

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration

Protein crystal presentation for synchrotron methods:acoustic techniques

This thesis describes the design, development and testing of novel acoustic mounting methods for the diffraction of protein crystals for structural biology. Sample environment and presentation is a challenging field set within the larger subject of protein crystallography. The needs of researchers to achieve the highest resolution data collection from difficult to fabricate samples, sits alongside the need for complex experimental conditions, where temperature, hydration and chemistry must be altered and controlled. A movement within the structural biology field away from obtaining structures at cryogenic temperatures, towards high resolution structures at room temperature, where proteins may still function, has been the driver to search for novel solutions. To approach this ‘solution space’ the following work draws on acoustic manipulation techniques, looking for self-assembly and non-contact manipulation. Thus for the first time acoustic standing wave crystal trapping and also acoustically induced rotation have been shown in situ to be viable for use with protein crystallography, a fundamental proof paving the way for new time resolved and high throughput methods. The methods investigated included both surface acoustic and bulk acoustic waves, looking at self-assembly and flow induced rotation. Each demonstration addresses a fundamental need within the automation of room temperature crystallography, demonstrating both the technique and quantifying the diffraction resolution for the first time. Through the completion of these novel experiments acoustic sample presentation has been proven viable. The demonstrated method does not require crystals be removed from crystallisation fluid before mounting (using the acoustic trapping method), thus enabling the application of secondary fluids and paving the way for a fully continuous and microfluidic technology. Moreover acoustic goniometry lends itself to the automated mounting and collection of small batch crystal data by removing the need for delicate spine mounting. Both methods constitute a significant extension in the ability of researchers to utilise non-contact methods to control and interact with their proteins and crystals at room temperature

Continuous focusing and separation of microparticles with acoustic and magnetic fields

Microfluidics enables a diverse range of manipulations (e.g., focusing, separating, trapping, and enriching) of micrometer-sized objects, and has played an increasingly important role for applications that involve single cell biology and the detection and diagnosis of diseases. In microfluidic devices, methods that are commonly used to manipulate cells or particles include the utilization of hydrodynamic effects and externally applied field gradients that induce forces on cells/particles, such as electrical fields, optical fields, magnetic fields, and acoustic fields. However, these conventional methods often involve complex designs or strongly depend on the properties of the flow medium or the interaction between the fluid and fluidic channels, so this dissertation aims to propose and demonstrate novel and low-cost techniques to fabricate microfluidic devices to separate microparticles with different sizes, materials and shapes by the optimized acoustic and magnetic fields. The first method is to utilize acoustic bubble-enhanced pinched flow for microparticle separation; the microfluidic separation of magnetic particles with soft magnetic microstructures is achieved in the second part; the third technique separates and focuses microparticles by multiphase ferrofluid flows; the fourth method realizes the fabrication and integration of microscale permanent magnets for particle separation in microfluidics; magnetic separation of microparticles by shape is proposed in the fifth technique. The methods demonstrated in this dissertation not only address some of the limitations of conventional microdevices, but also provide simple and efficient method for the separation of microparticles and biological cells with different sizes, materials and shapes, and will benefit practical microfluidic platforms concerning micron sized particles/cells --Abstract, page iv

Microscopic Particle Manipulation via Optoelectronic Devices

The optoelectronic tweezers (or optically induced dielectrophoresis (DEP)) have showed the ability to parallelly position a large number of colloidal microparticles without any template. The microparticles can be trapped and driven by the dielectrophoretic forces induced by the optical micropatterns in OET devices. In this chapter, the design and fabrication of flat optoelectronic devices (FOD) and hybrid optoelectronic device (HOD) are described. In the typical FOD, the manipulation modes including filtering, transporting, concentrating and focusing controlling regimes are experimentally demonstrated and analyzed. The controllable rotation of self-assembled microparticle chains in FOD has also been investigated, and a method incorporating the optically induced electrorotation (OER) and AC electroosmotic (ACEO) effects is numerically and experimentally implemented for manipulating microparticle chains. Based on the above research of FOD, a hybrid DEP microdevice HOD is conceptually and experimentally proposed. The HOD integrates with metallic microelectrode layer and the underneath photoconductive layer with projected optical virtual electrodes. FOD and HOD hybrid device enables the active driving, large-scale patterning and local position adjustment of microparticles. These techniques make up the shortcoming of low flexibility of traditional metallic microelectrodes and integrate the merits of both the metal electrode-induced and microimage-induced DEP techniques

Roadmap for optical tweezers

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMOptical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space explorationEuropean Commission (Horizon 2020, Project No. 812780