Gary Lee Thompson

Towards local electromechanical probing of cellular and biomolecular systems in a liquid environment

Electromechanical coupling is ubiquitous in biological systems, with examples ranging from simple piezoelectricity in calcified and connective tissues to voltage-gated ion channels, energy storage in mitochondria, and electromechanical activity in cardiac myocytes and outer hair cell stereocilia. Piezoresponse force microscopy (PFM) originally emerged as a technique to study electromechanical phenomena in ferroelectric materials, and in recent years has been employed to study a broad range of non-ferroelectric polar materials, including piezoelectric biomaterials. At the same time, the technique has been extended from ambient to liquid imaging on model ferroelectric systems. Here, we present results on local electromechanical probing of several model cellular and biomolecular systems, including insulin and lysozyme amyloid fibrils, breast adenocarcinoma cells, and bacteriorhodopsin in a liquid environment. The specific features of PFM operation in liquid are delineated and bottlenecks on the route towards nanometre-resolution electromechanical imaging of biological systems are identified.

Functional recognition imaging using artificial neural networks: applications to rapid cellular identification via broadband electromechanical response

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

Modeling Piezoresponse Force Microscopy for Low-Dimensional Material Characterization: Theory and Experiment

Piezoresponse force microscopy (PFM) is an atomic force microscopy-based approach utilized for measuring local properties of piezoelectric materials. The objective of this study is to propose a practical framework for simultaneous estimation of the local stiffness and piezoelectric properties of materials. For this, the governing equation of motion of a vertical PFM is derived at a given point on the sample. Using the expansion theorem, the governing ordinary differential equations of the system and their state-space representation are derived under applied external voltage. For the proof of the concept, the results obtained from both frequency and step responses of a PFM experiment are utilized to simultaneously identify the microcantilever parameters along with local spring constant and piezoelectric coefficient of a periodically poled lithium niobate sample. In this regard, a new parameter estimation strategy is developed for modal identification of system parameters under general frequency response. Results indicate good agreements between the identified model and the experimental data using the proposed modeling and identification framework. This method can be particularly applied for accurate characterization of mechanical and piezoelectric properties of biological species and cells.

Electromechanical and elastic probing of bacteria in a cell culture medium

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbeccos phosphate-buffered saline (DPBS) and Dulbeccos modified Eagles medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

Local plasma membrane permeabilization of living cells by nanosecond electric pulses using atomic force microscopy

Numerous studies provide evidence that nanosecond electric pulses (nsEPs) can trigger the formation of nanopores in the plasma membranes of cells. However, the biophysical mechanism responsible for nanopore formation is not well understood. In this study, we hypothesize that membrane damage induced by nsEPs is primarily dependent on the local molecular composition and mechanical strength of the plasma membrane. To test this hypothesis, we positioned metal-coated, nanoscale cantilever tips using an atomic force microscope (AFM) to deliver nsEPs to localized areas on the surface of the plasma membrane. We conducted computational modeling simulations to verify that the electric field provided by the nsEP is concentrated between the tip and the plasma membrane. The results show that we could effectively deliver nsEPs using the AFM tips at very low voltages. Using scanning electron microscopy we analyzed the tips after applying 10V over 5 seconds duration and found no damage to the tip or loss of platinum coating. As a proof of concept, we applied a 1 and 10V, 5 second pulse to HeLa cells causing large morphological changes. We also applied both a mechanical indention and 600ns electrical pulse stimulus and measured positive propidium ion uptake into the cytoplasm suggesting formation of membrane pores. In future studies, we plan to elucidate the effect that specific, local molecular structures and compositions have on efficacy of electroporation using the newly constructed nano-electrode system.

Detection of local stiffness and piezoelectric properties of materials via piezoresponse force microscopy

The objective of this study is to propose a practical framework for simultaneous estimation of the local stiffness and piezoelectric properties of materials via piezoresponse force microscopy (PFM). For this, the governing equation of motion of a vertical PFM is derived at a given point on the sample. Using the expansion theorem, the governing ordinary differential equations (ODEs) of the system and their state-space representation are derived under applied external voltage. For the proof of the concept, the results obtained from both frequency and step responses of a PFM experiment are utilized to simultaneously identify the microcantilever parameters along with local spring constant and piezoelectric coefficient of a Periodically Poled Lithium Niobate (PPLN) sample. In this regard, a new parameter estimation strategy is developed for modal identification of system parameters under general frequency response. Results indicate good agreements between the identified model and the experimental data using the proposed modeling and identification framework.

Stiffness and mass detection of nano layers using piezoresponse force microscopy

Piezoresponse force microscopy (PFM) is proposed in this article as a new technique for identification of elastically distributed thin layers on top of microcantilever sensors. Using the conventional actuation methods such as base excitation, the ratio of stiffness over the layer mass per unit length affects the resonant frequencies of the system as a single parameter. However, due to tip/sample elastic contact in PFM, these two parameters can be separately identified using the frequency shifts before and after attaching the layer. The concept is theoretically proven here using the modal analysis of the system. For practical verification, three gold-coated AFM microcantilevers were primarily tested for their initial resonant frequencies. The Focused Ion Beam (FIB) technique was then employed to deposit thin layers of Pt-based material in different configurations on the cantilevers’ surfaces. The microcantilevers were then reexamined for their new resonances, and the properties of the deposits were identified using a robust system identification procedure. Results indicate acceptable estimation of the cantilevers’ added mass and stiffness, making the technique suitable for detection of elastically distributed biological species.Copyright

Electromechanical imaging of biological systems with sub-10 nm resolution

Piezoelectricity in calcified and connective tissues has been known for over half a century following seminal works by Fukada. Traditionally, complex hierarchical structure of these materials, spanning the length scales from nanometers to millimeters, precluded quantitative studies, and hence elucidation of biological significance of biopiezoelectricity. Here, we demonstrate an approach for electromechanical imaging of structure of biological samples on the length scales from tens of microns to nanometers using piezoresponse force microscopy (PFM). In this method, intrinsic piezoelectricity of biopolymers such as proteins and polysaccharides is the basis for high-resolution imaging. Nanostructural imaging of a variety of protein-based materials, including tooth, antler, and cartilage is demonstrated. Visualization of protein fibrils with sub-10 nm spatial resolution in a human tooth is achieved. Given the near-ubiquitous presence of piezoelectricity in biological systems, PFM is suggested as a versatile tool for micro- and nanostructure imaging in both connective and calcified tissues. In the second part of the talk, I discuss recent advances in bioPFM, including imaging in liquid environments, resonance-enhanced PFM, and electrically shielded probes required to probe a broad range of electrophysiological processes from membrane flexoelectricity to cardiac miocyte activity.