Quantification of mechanical forces and physiological processes involved in pollen tube growth using microfluidics and microrobotics

Pollen tubes face many obstacles on their way to the ovule. They have to decide whether to navigate around cells or penetrate the cell wall and grow through it or even within it. Besides chemical sensing, which directs the pollen tubes on their path to the ovule, this involves mechanosensing to determine the optimal strategy in specific situations. Mechanical cues then need to be translated into physiological signals, which eventually lead to changes in the growth behavior of the pollen tube. To study these events, we have developed a system to directly quantify the forces involved in pollen tube navigation. We combined a lab-on-a-chip device with a microelectromechanical systems-based force sensor to mimic the pollen tube's journey from stigma to ovary in vitro. A force-sensing plate creates a mechanical obstacle for the pollen tube to either circumvent or attempt to penetrate while measuring the involved forces in real time. The change of growth behavior and intracellular signaling activities can be observed with a fluorescence microscope

Lab-On-Chip for Ex-Vivo study of morphogenesis of tip growing cells of pollen tube

The purpose of the thesis is to develop a microfluidic based lab-on-chip (LOC) platform providing an Ex-Vivotesting environment that is able to mimic certain aspects of the in vivo growth conditions of the pollen tube, a cellular protuberance formed by the male gametophyte in the flowering plants. The thesis focuses on design, fabrication, modeling and testing of various LOC devices for the study of static and dynamic behavior of pollen tubes in response to mechanical stimulation. TipChip, an LOC platform, was developed to advance both experimentation and phenotyping in cell tip growth research. The platform enabled simultaneous testing of multiple pollen tubes. Using TipChip, we were able to answer several outstanding questions regarding pollen tube biology. We found that contrary to other types of tip growing cells such as root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, we explored the effect of geometry of the microfluidic cell culture on pollen tube growth. We found that changing the width of the microfluidic channels does not have a significant effect on the pollen tube growth rate, while the growth rate was increased by increasing microchannel depth. We modified the original TipChip design to ascertain identical growth conditions for sequentiallyarranged pollen tubes and to ensure even distribution of entrapment probabilities for all microchannels. The effect of different dimensions of the microfluidic network on cell trapping probability was assessed using computational fluid dynamics and verified by experimental testing. The design was optimized based on trapping probability and uniformity of fluid flow conditions within the microchannels. This thesis also presents a novel method of fabricating a high aspect ratio horizontal PDMS microcantilever-based flow sensor integrated into a microfluidic device. The performance of the flow sensor was tested by introducing various flow rates into the microfluidic device and measuring the deflection of the cantilever’s tip using an optical microscope. The thesis addresses the quantification of cellular growth force of Camellia pollen tip growing cells using FlexChip, a flexure integrated LOC on polymer. We quantified the force that pollen tube is able to exert using a microfluidic lab-on-a-chip device integrated with flexural structure. The pollen grain is trapped in the microfluidic network and the growing tube is guided against a flexible microstructure that is monolithically integrated within the microfluidic chip. The invasive growth force of growing pollen tube was calculated from the maximal bending of microstructure modelled by Finite Element Analysis (FEA). Furthermore, the effect of the mechanical obstacle on the pollen tube's growth dynamics was assessed by quantifying the shift in the peak frequency characterizing the oscillatory behavior of the pollen tube growth rate. Our detailed analysis of the pollen tube growth dynamic before and during the contact with microcantilever revealed that pollen tube growth rate was reduced by 44% during the contact with the microcantilever. Moreover, the peak of oscillation frequency of pollen tube growth rate was reduced more dramatically by 70-75%. This suggests that the pollen tube actively changes its growth pattern to cope with the mechanical obstacle. Our findings in this thesis are novel in terms of pollen biology, and we believe insights from this research will lead to a better understanding of morphogenesis of a kind of tip growing cells, namely, pollen tube

Plant hydration dynamics: measurement and uptake pathways

Transpiration accounts for most terrestrial water fluxes, and agriculture uses most of the water managed by humans. Transpiration is tightly regulated by plants, so climate models and irrigation water use efficiency could be improved by understanding how plants regulate water status. In this work, I address questions that are relevant to our understanding of plant hydration dynamics: (1) Can foliar water uptake (FWU) restore leaf hydraulic conductance (Kleaf) lost due to dehydration? (2) Is embolism refilling involved in FWU-induced hydraulic recovery? (3) Can plant water status be measured by uniaxial compression of the leaf lamina? Many plants are able to access atmospheric water through FWU; however, the physiological consequences of FWU are unclear. While FWU represents a small water flux, it may play a role in restoring hydraulic conductivity lost during dehydration. My results showed that FWU can restore Kleaf in Avicennia marina lost during dehydration. While hydraulic recovery retraced the same path observed during dehydration-dependent loss of Kleaf, a reduced ability for FWU impaired Kleaf recovery under severe dehydration. Most of the resistance to FWU was located in the leaf surface. I conclude that FWU may play a role in the maintenance of shoot hydraulic function during changing water status. Plants living in saline environments experience constant xylem tension. Under these conditions, it is unclear how embolism refilling can take place. Using micro-CT imaging, I imaged Avicennia marina twigs in a dehydrated state and 4-48 h after wetting the twig surface. Emboli were present in the stem and leaves in the dehydrated state. Stem emboli were likely caused by cutting, while leaf emboli were likely caused by dehydration. Emboli in stems and leaves refilled with water after wetting, taking up to 48 h in the process, which is slower than the documented FWU rehydration kinetics. Possibly, refilling was facilitated by a vascular constriction at the stem-petiole junction and/or by loading of inorganic solutes into xylem vessels. My results substantiate that FWU is an important source of water for this widespread mangrove species; however, differences between field and experimental conditions currently preclude extrapolating these results to natural settings. Turgor is an essential indicator of plant water status; however, turgor measurements are not routine. Turgor can be measured by localised compression of cells or tissues, but an accessible method to perform these measurements is lacking. I hypothesized that leaf turgor pressure can be monitored by uniaxially compressing the leaf lamina and by measuring the stress under a constrained thickness ('stress relaxation', SR); and that leaf water content can be monitoring by measuring the thickness of leaves compressed under a constant force ('constant stress', CS). Using a c. US$300 leaf squeeze-flow rheometer, I showed that uniaxial compression provides accurate measurement of plant water status with high temporal resolution at low cost. Experimental results and a simple hydrostatic equilibrium model indicate that the stationary bulk modulus during compression is largely determined by the bulk osmotic pressure. Leaf squeeze-flow rheometry is presented as a novel, automatable and potentially standard method to quantify plant water status

A Novel Propeller Design for Micro-Swimming robot

The applications of a micro-swimming robot such as minimally invasive surgery, liquid pipeline robot etc. are widespread in recent years. The potential application fields are so inspiring, and it is becoming more and more achievable with the development of microbiology and Micro-Electro-Mechanical Systems (MEMS). The aim of this study is to improve the performance of micro-swimming robot through redesign the structure. To achieve the aim, this study reviewed all of the modelling methods of low Reynolds number flow including Resistive-force Theory (RFT), Slender Body Theory (SBT), and Immersed Boundary Method (IBM) etc. The swimming model with these methods has been analysed. Various aspects e.g. hydrodynamic interaction, design, development, optimisation and numerical methods from the previous researches have been studied. Based on the previous design of helix propeller for micro-swimmer, this study has proposed a novel propeller design for a micro-swimming robot which can improve the velocity with simplified propulsion structure. This design has adapted the coaxial symmetric double helix to improve the performance of propulsion and to increase stability. The central lines of two helical tails overlap completely to form a double helix structure, and its tail radial force is balanced with the same direction and can produce a stable axial motion. The verification of this design is conducted using two case studies. The first one is a pipe inspection robot which is in mm scale and swims in high viscosity flow that satisfies the low Reynolds number flow condition. Both simulation and experiment analysis are conducted for this case study. A cross-development method is adopted for the simulation analysis and prototype development. The experiment conditions are set up based on the simulation conditions. The conclusion from the analysis of simulation results gives suggestions to improve design and fabrication for the prototype. Some five revisions of simulation and four revisions of the prototype have been completed. The second case study is the human blood vessel robot. For the limitations of fabrication technology, only simulation is conducted, and the result is compared with previous researches. The results show that the proposed propeller design can improve velocity performance significantly. The main outcomes of this study are the design of a micro-swimming robot with higher velocity performance and the validation from both simulation and experiment

A microrobotic system for simultaneous measurement of turgor pressure and cell-wall elasticity of individual growing plant cells

ISSN:2377-376

A Microrobotic System for Simultaneous Measurement of Turgor Pressure and Cell-Wall Elasticity of Individual Growing Plant Cells

Plant growth and morphogenesis is directed by cell division and the expansion of individual cells. How the tightly controlled process of cell expansion is regulated is poorly understood. We introduce a microrobotic platform able to separately measure the turgor pressure and cell wall elasticity of individual growing, turgid cells by combining microindentation with cell compression experiments. The system independently controls two indenters with geometries at different scales. Indentation measurements are performed automatically by deforming the cells with indenters with a spatial resolution in the nanometer range while recording force and displacement. The dual-indentation technique offers a noninvasive, high-throughput method to characterize the cytomechanics of single turgid cells by separately measuring elasticity and turgor pressure. In this way, the expansion regulation of growing cells can be investigated, as demonstrated here using Lilium longiflorum pollen tubes as an example