Genetic material manipulation and modification by optical trapping and Nanosurgery-A perspective

Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers, in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presente

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

Passive and Active Microrheology for Biomedical Systems

Microrheology encompasses a range of methods to measure the mechanical properties of soft materials. By characterizing the motion of embedded microscopic particles, microrheology extends the probing length scale and frequency range of conventional bulk rheology. Microrheology can be characterized into either passive or active methods based on the driving force exerted on probe particles. Tracer particles are driven by thermal energy in passive methods, applying minimal deformation to the assessed medium. In active techniques, particles are manipulated by an external force, most commonly produced through optical and magnetic fields. Small-scale rheology holds significant advantages over conventional bulk rheology, such as eliminating the need for large sample sizes, the ability to probe fragile materials non-destructively, and a wider probing frequency range. More importantly, some microrheological techniques can obtain spatiotemporal information of local microenvironments and accurately describe the heterogeneity of structurally complex fluids. Recently, there has been significant growth in using these minimally invasive techniques to investigate a wide range of biomedical systems both in vitro and in vivo . Here, we review the latest applications and advancements of microrheology in mammalian cells, tissues, and biofluids and discuss the current challenges and potential future advances on the horizon

The effects of cold arm width and metal deposition on the performance of a U-Beam electrothermal MEMS microgripper for biomedical applications

Microelectromechanical systems (MEMS) have established themselves within various fields dominated by high-precision micromanipulation, with the most distinguished sectors being the microassembly, micromanufacturing and biomedical ones. This paper presents a horizontal electrothermally actuated 'hot and cold arm' microgripper design to be used for the deformability study of human red blood cells (RBCs). In this study, the width and layer composition of the cold arm are varied to investigate the effects of dimensional and material variation of the cold arm on the resulting temperature distribution, and ultimately on the achieved lateral displacement at the microgripper arm tips. The cold arm widths investigated are 14 μm, 30 μm, 55 μm, 70 μm and 100 μm. A gold layer with a thin chromium adhesion promoter layer is deposited on the top surface of each of these cold arms to study its effect on the performance of the microgripper. The resultant ten microgripper design variants are fabricated using a commercially available MEMS fabrication technology known as a silicon-on-insulator multi-user MEMS process (SOIMUMPs)TM. This process results in an overhanging 25 μm thick single crystal silicon microgripper structure having a low aspect ratio (width:thickness) value compared to surface micromachined structures where structural thicknesses are of the order of 2 μm. Finite element analysis was used to numerically model the microgripper structures and coupled electrothermomechanical simulations were implemented in CoventorWare ®. The numerical simulations took into account the temperature dependency of the coefficient of thermal expansion, the thermal conductivity and the electrical conductivity properties in order to achieve more reliable results. The fabricated microgrippers were actuated under atmospheric pressure and the experimental results achieved through optical microscopy studies conformed with those predicted by the numerical models. The gap opening and the temperature rise at the cell gripping zone were also compared for the different microgripper structures in this work, with the aim of identifying an optimal microgripper design for the deformability characterisation of RBCs.peer-reviewe

Doctor of Philosophy

dissertationMicrofluidics is an emerging field that deals with the technology and science of manipulation of fluid in microchannels. Since its birth in the 1990s, it has now gradually matured into an enabling technology, like microelectronics and software engineering. A majority of current applications of microfluidics are in life sciences. Polydimethylsiloxane (PDMS) is a soft elastomer and a popular material for fabricating microfluidic devices. This is due to PDMS's unique set of material properties and low cost. Furthermore, the unique mechanical properties of thin PDMS layers/membranes (< 200 µm) can be used to increase the functionality of PDMS-based microfluidic systems. In this presentation, three unique neuroscience applications of PDMS-based microfluidic devices are presented. The working principle behind each of these devices depends on the unique properties of thin PDMS layers. In the first project a fabrication protocol was developed to stack 30 patterned 10-um thick PDMS layers on top of each other without any trapped air bubbles or wrinkles. Each PDMS layer was patterned by spin-coating uncured PDMS on a photolithographic micromold at very high spin speeds and thermally curing the layer later. The layer stacking procedure was done manually using no specialized tools and did not cause any layer deformation to inhibit functionality. This fabrication protocol was used to develop the first ever microfluidic Magnetic Resonance Imaging Phantom to stimulate brain white matter. In the second project, laser ablation was used to rapidly prototype micromolds and by using these micromolds a unique fabrication protocol was developed and characterized to build microvalve arrays (consisting of 100s of microvalves) without access to any cleanroom facility. This was achieved by manipulating the stiffness of thin PDMS layers that are inherent part of pneumatic microvalves. These microvalve arrays were used to build a microfluidic platform for manipulation of C. elegans (a type of a small round worm), which are used extensively for neuronal behavioral analysis. In the last project using similar fabrication techniques (as described in the second project) microfluidic genotyping devices are developed for zebrafish embryos that are less than 2 days old. The unique advantage of the microfluidic zebrafish genotyping devices is that they enable researchers to collect genetic material (for genotyping) from a zebrafish embryo (1 to 2 days old) without causing any harm to its health. This capability is not possible with any other model multicellular organism to date. The working principle behind one of the presented genotyping devices depends on the controlled actuation of PDMS membranes

Cell Mechanics in Physiology: A Force Based Approach

All biological systems rely on complex interactions with their external and internal environments where the key factors are force sensing and force generation. These systems are highly dynamic, and recent studies have shown that it is the control and maintenance of these interactions that are essential for normal functioning. Appreciation of these roles has led to a revolution in instrumentation and techniques to study and model mechanical interaction at all length and time scales in biology. The work presented here is one such effort, utilizing a magnetics based force system to study and understand the mechanisms of cell mechanics and their role in mucuciliary clearance in the lung and in cancer cell invasion and metastasis. I first introduce the instrumentation and describe basic rheological concepts that govern the study of cell mechanics. I then report on the application of this system to study the force generation and dynamics of airway cilia. The bulk of the work is focussed on the role of cytoskeleton mechanics in cancer. I present our results which show the remarkable relationship between the cell's mechanical properties and its metastatic potential. Finally, I report on a novel pathway which is responsible for force mediated sensing in cells and show that this pathway is deregulated in cancer. These results have strong implications on the potential of stiffness and force sensing pathways as novel cancer therapeutic targets

Techniques to stimulate and interrogate cell–cell adhesion mechanics

Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechanotransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These studies leverage technical advances in devices and systems interfacing with living cells through cell–extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experimental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers

The development of optical nanomachines for studying molecules : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Mechatronics Engineering at Massey University, Palmerston North, New Zealand

Chapter 3 is ©2020 IEEE. Accepted manuscript is reprinted, with permission, from 2020 IEEE/ASME International Conference on Advanced Intelligent Mechatronics (AIM). Chapter 5 is ©2022 IEEE. Accepted manuscript is reprinted, with permission, from 2022 International Conference on Manipulation, Automation and Robotics at Small Scales (MARSS).Optical tweezers have been used for a number of applications since their invention by Arthur Ashkin in 1986, and are particularly useful for biological and biophysical studies due to their exceptionally high spatial and force-based resolution. The same intense laser focus that allows light to be used as a tool for micro-nanoscale manipulation also has the potential to damage the objects being studied, and the extremely high force resolution is coupled with the limitation of very low forces. There is potential to overcome these drawbacks of optical manipulation through making use of another laser based technique: two-photon absorption polymerisation (TPAP). This thesis has brought these together to demonstrate the uses of optical nanomachines as helpful tools for optical tweezer studies. The project was highly interdisciplinary, concerning the intersection of optical trapping, 3D micromachine design and development, and DNA stretching. The thesis was based around the strategy of first developing microrobots and demonstrating their manipulation using optical tweezers, then adjusting the design for specific applications. Microlevers were developed for lever-assisted DNA stretching and amplification of optical forces. The influence of design features and TPAP parameters on microlever functionality was investigated; particularly the influence of overlapping area and presence of supports, and the effects of differently shaped "trapping handles". These features were important as lever functionality was tested in solutions of different ionic strength, and stable trapping of the levers was required for force amplification. DNA stretching was chosen as a target application for distanced-application of optical forces due to its status as a well-known and characterised example of single-molecule studies with optical tweezers. Amplification of optical forces was also seen as an application that could demonstrate the utility of optical micromachines, and microlevers with a 2:1 lever arm ratio were developed to produce consistent, two-fold amplification of optical forces, in a first for unsupported, pin-jointed optical microrobotics. It is hoped that in the future fully-remote, micromachine-assisted studies will extend optical tweezer studies of laser-sensitive subjects, as well as increasing the forces that can be applied, and the results obtained in this thesis are encouraging. All in all, the thesis confirms the potential of optical micromachines for aiding studies using optical tweezers, and demonstrates concrete progress in both design and application

A SINGLE CELL PAIR MECHANICAL INTERROGATION PLATFORM TO STUDY CELL-CELL ADHESION MECHANICS

Cell-cell adhesion complexes are macromolecular adhesive organelles that integrate cells into tissues. Perturbations of the cell-cell adhesion structure or relatedmechanotransduction pathways lead to pathological conditions such as skin and heart diseases, arthritis, and cancer. Mechanical stretching has been used to stimulate the mechanotransduction process originating from the cell-cell adhesion and cell-extracellular matrix (ECM) complexes. The current techniques, however, have limitations on their ability to measure the cell-cell adhesion force directly and quantitatively. These methods use a monolayer of cells, which makes it impossible to quantify the forces within a single cell-cell adhesion complex. Other methods using single cells or cell pairs rely on cell-ECM adhesion to find the cell-cell adhesion forces and consequently, they indirectly measure the junctional forces. In the current study, we designed and developed a single cell-cell adhesion interrogation and stimulation platform based on nanofabricated polymeric structures. The platform employs microstructures fabricated from biocompatible materials using two photon polymerization (TPP), a process that enables direct 3D structure writing with nanometer precision. The microdevice allows a pair of epithelial cells to form a mature cell junction. The single matured cell junction is stretched with controlled strain until cell-cell junction ruptures while the forces within the cell-junction-cell system are recorded. Using this platform, we have conducted mechanical characterization of a single cell junction with strain-stress analysis. The strain dependency of the junction has been investigated through the stretch test with four different strain rates. The results showed that the junction behaves in a strain-rate dependent manner, where high strain-rates lead to decreased viscosity property, a characteristic for a shear-thinning viscoelastic material. This also confirms our hypothesis that strain-rate plays an important role in the cell mechanical behavior, particularly the cytoskeleton dominant cell mechanics. The maturation of this technology can pave the way for the in situ investigation of mechano-chemical signaling pathways mediated by cell-cell junctions and potentially reveal novel disease mechanisms in which defects in cell-cell adhesion play a significant role in the disease pathology. Advisor: Ruiguo Yan