PLANNING FOR AUTOMATED OPTICAL MICROMANIPULATION OF BIOLOGICAL CELLS

Optical tweezers (OT) can be viewed as a robot that uses a highly focused laser beam for precise manipulation of biological objects and dielectric beads at micro-scale. Using holographic optical tweezers (HOT) multiple optical traps can be created to allow several operations in parallel. Moreover, due to the non-contact nature of manipulation OT can be potentially integrated with other manipulation techniques (e.g. microfluidics, acoustics, magnetics etc.) to ensure its high throughput. However, biological manipulation using OT suffers from two serious drawbacks: (1) slow manipulation due to manual operation and (2) severe effects on cell viability due to direct exposure of laser. This dissertation explores the problem of autonomous OT based cell manipulation in the light of addressing the two aforementioned limitations. Microfluidic devices are well suited for the study of biological objects because of their high throughput. Integrating microfluidics with OT provides precise position control as well as high throughput. An automated, physics-aware, planning approach is developed for fast transport of cells in OT assisted microfluidic chambers. The heuristic based planner employs a specific cost function for searching over a novel state-action space representation. The effectiveness of the planning algorithm is demonstrated using both simulation and physical experiments in microfluidic-optical tweezers hybrid manipulation setup. An indirect manipulation approach is developed for preventing cells from high intensity laser. Optically trapped inert microspheres are used for manipulating cells indirectly either by gripping or pushing. A novel planning and control approach is devised to automate the indirect manipulation of cells. The planning algorithm takes the motion constraints of the gripper or pushing formation into account to minimize the manipulation time. Two different types of cells (Saccharomyces cerevisiae and Dictyostelium discoideum) are manipulated to demonstrate the effectiveness of the indirect manipulation approach

Preparation of Tissues and Heterogeneous Cellular Samples for Single-Cell Analysis

While sample preparation techniques for the chemical and biochemical analysis of tissues are fairly well advanced, the preparation of complex, heterogenous samples for single-cell analysis can be difficult and challenging. Nevertheless, there is growing interest in preparing complex cellular samples, particularly tissues, for analysis via single-cell resolution techniques such as single-cell sequencing or flow cytometry. Recent microfluidic tissue dissociation approaches have helped to expedite the preparation of single cells from tissues through the use of optimized, controlled mechanical forces. Cell sorting and selective cellular recovery from heterogenous samples have also gained traction in biosensors, microfluidic systems, and other diagnostic devices. Together, these recent developments in tissue disaggregation and targeted cellular retrieval have contributed to the development of increasingly streamlined sample preparation workflows for single-cell analysis technologies, which minimize equipment requirements, enable lower processing times and costs, and pave the way for high-throughput, automated technologies. In this chapter, we survey recent developments and emerging trends in this field

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

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

Internalisation of biophotonic techniques : transfection, injection and thermometry

Single cell manipulation can offer great insights into the whole of an organism, the rapidly growing -omics fields are illustrating the heterogeneity that can be found within cell populations and where these subtle differences may be exploited, from fundamental knowledge to diagnostics and therapeutics. The cutting edge of this single cell work requires the application of interdisciplinary research to fully exploit the boundaries being pushed. Biophotonics is one such body of interdisciplinary research, employing light to manipulate biological samples. This work seeks to make use biophotonic techniques as analogues for conventional biological methods. High throughput raster scan photoporation is utilised for attempted transfection, multiple trap optical tweezers are used in an attempt to optically drive mechanical injection of cells and the thermal impact of these optical tweezers, which require high energy densities to confine particles, is tested, via the exploitation of the temperature sensitive emission of quantum dot nanoparticles

An aptamer-based sensing platform for luteinising hormone pulsatility measurement

Normal fertility in human involves highly orchestrated communication across the hypothalamic-pituitary-gonadal (HPG) axis. The pulsatile release of Luteinising Hormone (LH) is a critical element for downstream regulation of sex steroid hormone synthesis and the production of mature eggs. Changes in LH pulsatile pattern have been linked to hypothalamic dysfunction, resulting in multiple reproductive and growth disorders including Polycystic Ovary Syndrome (PCOS), Hypothalamic Amenorrhea (HA), and delayed/precocious puberty. Therefore, assessing the pulsatility of LH is important not only for academic investigation of infertility, but also for clinical decisions and monitoring of treatment. However, there is currently no clinically available tool for measuring human LH pulsatility. The immunoassay system is expensive and requires large volumes of patient blood, limiting its application for LH pulsatility monitoring. In this thesis, I propose a novel method using aptamer-enabled sensing technology to develop a device platform to measure LH pulsatility. I first generated a novel aptamer binding molecule against LH by a nitrocellulose membrane-based in vitro selection then characterised its high affinity and specific binding properties by multiple biophysical/chemical methods. I then developed a sensitive electrochemical-based detection method using this aptamer. The principal mechanism is that structure switching upon binding is associated with the electron transfer rate changes of the MB redox label. I then customised this assay to numerous device platforms under our rapid prototyping strategy including 96 well automated platform, continuous sensing platform and chip-based multiple electrode platform. The best-performing device was found to be the AELECAP (Automated ELEctroChemical Aptamer Platform) – a 96-well plate based automatic micro-wire sensing platform capable of measuring a series of low volume luteinising hormone within a short time. Clinical samples were evaluated using AELECAP. A series of clinical samples were measured including LH pulsatility profile of menopause female (high LH amplitude), normal female/male (normal LH amplitude) and female with hypothalamic amenorrhea (no LH pulsatility). Total patient numbers were 12 of each type, with 50 blood samples collected every 10 mins in 8 hours. Results showed that the system can distinguish LH pulsatile pattern among the cohorts and pulsatility profiles were consistent with the result measured by clinical assays. AELECAP shows high potential as a novel approach for clinical aptamer-based sensing. AELECAP competes with current automated immunometric assays system with lower costs, lower reagent use, and a simpler setup. There is potential for this approach to be further developed as a tool for infertility research and to assist clinicians in personalised treatment with hormonal therapy.Open Acces

Tools for single cell proteomics

Despite recent advances that offer control of single cells, in terms of manipulation and sorting and the ability to measure gene expression, the need to measure protein copy number remains unmet. Measuring protein copy number in single cells and related quantities such as levels of phosphorylation and protein-protein interaction is the basis of single cell proteomics. A technology platform to undertake the analysis of protein copy number from single cells has been developed. The approach described is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by mechanical shearing caused by laser-induced microcavitation; and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. This demonstrates the ability count protein copy number from single cells in a manner which could be applied in principle to any set of proteins and for any cell type without the need for genetic engineering. Metabolism can undergo alteration in diseases such as cancer and heart failure and also as cells differentiate during development. In order to assess how it may inform a proteomic measurement, multidimensional two-photon fluorescence metabolic imaging is conducted on a cultured cancer cell line, primary adult rat cardiomyocytes and human embryonic stem cells. By measuring the parameters of fluorescence such as intensity and lifetime of the autofluorescent metabolic co-factors NADH and FAD, it was found to be possible to contrast cells under various conditions and metabolic stimuli. In particular, human embryonic stem cells were able to be contrasted at 3 stages of development as they underwent differentiation into embryonic stem cell derived cardiomyocytes. Metabolic imaging provides a non-destructive method to monitor cellular metabolic activity with high resolution. This is complimentary to the single cell proteomic platform and the convergence of both techniques holds promise in future investigations into how metabolism influences cell function and the proteome in development and disease

複数の微小流を用いたマイクロマニピュレーション

電気通信大学201

3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis

The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows: 1) COMSOL Multiphysics® was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated. 2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 µm channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 μm to 334 μm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 μm, each standard 20 μL reaction could produce ~10⁴ droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3×10³ target copies or cells per μL, and the detection limit is 0.1 copies or cells per μL. 3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations. 4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link. In summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.</p