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

REGULATION OF SHAPE DYNAMICS AND ACTIN POLYMERIZATION DURING COLLECTIVE CELL MIGRATION

This thesis aims to understand how cells coordinate their motion during collective migration. As previously shown, the motion of individually migrating cells is governed by wave-like cell shape dynamics. The mechanisms that regulate these dynamic behaviors in response to extracellular environment remain largely unclear. I applied shape dynamics analysis to Dictyostelium cells migrating in pairs and in multicellular streams and found that wave-like membrane protrusions are highly coupled between touching cells. I further characterized cell motion by using principle component analysis (PCA) to decompose complex cell shape changes into a serial shape change modes, from which I found that streaming cells exhibit localized anterior protrusion, termed front narrowing, to facilitate cell-cell coupling. I next explored cytoskeleton-based mechanisms of cell-cell coupling by measuring the dynamics of actin polymerization. Actin polymerization waves observed in individual cells were significantly suppressed in multicellular streams. Streaming cells exclusively produced F-actin at cell-cell contact regions, especially at cell fronts. I demonstrated that such restricted actin polymerization is associated with cell-cell coupling, as reducing actin polymerization with Latrunculin A leads to the assembly of F-actin at the side of streams, the decrease of front narrowing, and the decoupling of protrusion waves. My studies also suggest that collective migration is guided by cell-surface interactions. I examined the aggregation of Dictyostelim cells under distinct conditions and found that both chemical compositions of surfaces and surface-adhesion defects in cells result in altered collective migration patterns. I also investigated the shape dynamics of cells suspended on PEG-coated surfaces, which showed that coupling of protrusion waves disappears on touching suspended cells. These observations indicate that collective migration requires a balance between cell-cell and cell-surface adhesions. I hypothesized such a balance is reached via the regulation of cytoskeleton. Indeed, I found cells actively regulate cytoskeleton to retain optimal cell-surface adhesions on varying surfaces, and cells lacking the link between actin and surfaces (talin A) could not retain the optimal adhesions. On the other hand, suspended cells exhibited enhanced actin filament assembly on the periphery of cell groups instead of in cell-cell contact regions, which facilitates their aggregation in a clumping fashion

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

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

Roadmap for optical tweezers

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 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 exploration.journal articl

Visual Computing Tools for Studying Micro-scale Diffusion

In this dissertation, we present novel visual computing tools and techniques to facilitate the exploration, simulation, and visualization of micro-scale diffusion. Our research builds upon the latest advances in visualization, high-performance computing, medical imaging, and human perception. We validate our research using the driving applications of nano-assembly and diffusion kurtosis imaging (DKI). In both of these applications, diffusion plays a central role. In the former it mediates the process of transporting micron-sized particles through moving lasers, and in the latter it conveys brain micro-geometry. Nanocomponent-based devices, such as bio-sensors, electronic components, photonic devices, solar cells, and batteries, are expected to revolutionize health care, energy, communications, and the computing industry. However, in order to build such useful devices, nanoscale components need to be properly assembled together. We have developed a hybrid CPU/GPU-based computing tool to understand complex interactions between lasers, optical beads, and the suspension medium. We demonstrate how a high-performance visual computing tool can be used to accelerate an optical tweezers simulation to compute the force applied by a laser onto micro particles and study shadowing (refraction) behavior. This represents the first steps toward building a real-time nano-assembly planning system. A challenge in building such a system, however, is that optical tweezers systems typically lack stereo depth cues. We have developed a visual tool to provide an enhanced perception of a scene's 3D structure using the kinetic depth effect. The design of our tool has been informed by user studies of stereo perception using the kinetic-depth effect on monocular displays. Diffusion kurtosis imaging is gaining rapid adoption in the medical imaging community due to its ability to measure the non-Gaussian property of water diffusion in biological tissues. Compared with the traditional diffusion tensor imaging (DTI), DKI can provide additional details about the underlying microstructural characteristics of neural tissues. It has shown promising results in studies on changes in gray matter and mild traumatic brain injuries, where DTI is often found to be inadequate. However, the highly detailed spatio-angular fields in DKI datasets present a special challenge for visualization. Traditional techniques that use glyphs are often inadequate for expressing subtle changes in the DKI fields. In this dissertation, we outline a systematic way to manage, analyze, and visualize spatio-angular fields using spherical harmonics lighting functions to facilitate insights into the micro-structural properties of the brain

Investigating the mechanics of tumour: from single cell mechanics to the biophysical interplay between cells and ECM

This PhD project has focused the attention on the mechanical characterization of cancer cells and their surroundings. It is well known that the mechanical properties of cells and extracellular matrix (ECM), especially stiffness, play an important role in many biological processes such as cell growth, migration, division and differentiation. The pathological state of a cell implicates the alteration of the cytoskeletal structure and, consequently, of its functions, determining a variation of cell and ECM mechanical properties. In particular, the aim of this work is to investigate how cancer progression changes cell and ECM mechanical properties in vitro and ex vivo conditions. In the first experimental studies, particle tracking microrheology and Atomic Force Microscopy (AFM) techniques were performed to compare the mechanical properties of murine normal and virus-transformed cell lines cultured on glass. The first goal of the work was the identification of several biophysical parameters to discriminate between tumour and healthy cells. They have been useful to understand how virus transformation influence cell physiological processes and mechanical properties and, as a consequence, to identify the existence of a relationship between biological functions and cell mechanics. We observed that the effects of virus induced-transformation are the intensification of cell proliferation, the enhanced capability of transformed cells to migrate, the reduced adhesion capability, the reduction of cell cytoskeletal organization and the increased cell deformability. Successively, taking into account the results collected on the single murine cells, we moved to the characterization of human lung cells with different metastatic potential. Also in this case, combining the analyses of phenotypic characteristics and the biophysical properties of the cells, in particular elasticity, we were able to discriminate benign from cancer cells and, among them, to distinguish the grade of aggressiveness. Thus, we achieved the first milestone of this work with the definition of a new and accurate biomarker of cell metastatic potential. The second goal of the work concerned the investigation of the crosstalk between cancer cells and the surrounding ECM, through the study of ex vivo human biopsy tissues, removed from patients affected by lung adenocarcinoma. To this aim a new technique, based on multiple particle tracking (MPT) has been developed. To perform, at the same time, the mechanical classification of cells and ECM of each sample and a comparison with the healthy equivalent for the entire pool of patients, the ECM structure and morphology of cancer and healthy tissues were investigated and compared. Moreover, results and mechanical phenotypes were correlated to the stage and the grade of cancer, previously classified by the classic immunodiagnostic method. The cancerous transformation of tissues had a remarkable effect on the dynamics of the tracer beads and contributes a sort of symmetric modification of the mechanical properties of the cells and ECM. Indeed, compared to the healthy tissues, particles introduced into the cells of adenocarcinoma tissues increase their motion. Otherwise, unlike healthy tissues, the reduced motion of the beads probing the surrounding ECM suggests that cell in tumour tissues reside in a stiffer matrix. These increased mechanical properties of ECM are associated to an enhancement of collagen cross-linking, also confirmed through the structural and morphological analyses of tissue biopsies. The obtained mechanical properties of cells and their surrounding ECM from MPT represents a reliable indicator of the malignant transformation process and we believe that it can be used in combination with the classical immunohistochemistry-based diagnostic tools to obtain a more effective and precise diagnosis of the cancer

An investigation into the effects of substrate properties on the mechanics of corneal epithelial cells

Cells respond to mechanical changes in their extracellular environment, as reflected by various cell behaviours and observed through changes in the tissue biomechanics. Types of cell behaviour that are regulated by mechanical cues in the microenvironment of the cell are cell spreading, migration, proliferation and differentiation. Cell migration is a key part of many biological processes including corneal wound repair. Changes in the biomechanical properties of the cornea can be induced by refractive and therapeutic treatments and also by diseases of the eye or other illnesses. A Rabbit Corneal Epithelial (RCE) cell line was used to study cell mechanics and cell migration. Polydimethylsiloxane (PDMS), a biocompatible silicone elastomer, was used as a substrate to culture RCE cells. In order to promote cell attachment and growth, the hydrophilicity of the PDMS surface was increased by treating it with oxygen-rich cold atmospheric pressure plasma, which was confirmed by surface characterisation techniques. Cell attachment and growth studies over time comparing plasma and non-plasma treated PDMS showed an increase in RCE cell growth and area coverage on plasma treated PDMS. [Continues.

Optical trapping: optical interferometric metrology and nanophotonics

The two main themes in this thesis are the implementation of interference methods with optically trapped particles for measurements of position and optical phase (optical interferometric metrology) and the optical manipulation of nanoparticles for studies in the assembly of nanostructures, nanoscale heating and nonlinear optics (nanophotonics). The first part of the thesis (chapter 1, 2) provides an introductory overview to optical trapping and describes the basic experimental instrument used in the thesis respectively. The second part of the thesis (chapters 3 to 5) investigates the use of optical interferometric patterns of the diffracting light fields from optically trapped microparticles for three types of measurements: calibrating particle positions in an optical trap, determining the stiffness of an optical trap and measuring the change in phase or coherence of a given light field. The third part of the thesis (chapters 6 to 8) studies the interactions between optical traps and nanoparticles in three separate experiments: the optical manipulation of dielectric enhanced semiconductor nanoparticles, heating of optically trapped gold nanoparticles and collective optical response from an ensemble of optically trapped dielectric nanoparticles