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Advanced Nanofabrication and Microscopy Methods for Multidimensional Nanoscale Imaging and Characterization of Biological Materials
thesis
posted on 2017-05-01, 23:31 authored by VahidReza AdinehThe building
blocks of the basic unit of all known living organisms, i.e. cells, consist of
minute nanoscale particles such as DNA, ribosome and others. Developments in
cancer therapies, antibiotics and advanced drug delivery systems significantly
rely on the capability in acquiring chemical and structural information from
biological materials, particularly biological cells in three-dimensional
nanoscale resolution. Thus, advanced nanofabrication and microscopy methods to
characterize biological materials at the nanoscale in three-dimensional (3D)
resolution are highly expected for biomedical research. However, when it comes
to the application of advanced nanofabrication and microscopy methods in
multidimensional nanoscale imaging and characterization of biological
materials, the following two aspects need to be further explored:
(1) Methods and protocols to explore the biological systems of size micrometre scale with nanometer resolution, including the capabilities to acquire mechanical and chemical information from interior regions of large biological structures.
(2) 3D nanoscale chemical mapping of biological materials, particularly biological cells.
The 3D data are essential to understanding the biological and cellular structure, while at the same time nanoscale resolution is necessary to follow the thinnest biological and cellular processes and to identify building blocks of biological and cellular structures such as RNA, DNA, and associated minute proteins. Thus, the principal purpose of this PhD research is divided into two parts: (1) to devise a prototype platform to investigate mechanical and chemical properties of interior regions of large biomechanical systems at nanometer scale, and (2) to develop an optimal strategy based on the advanced nanofabrication techniques for the applications of novel microscopy methods on 3D near-atomic chemical composition and structural characterization of biological materials, particularly biological cells.
In the first part of this PhD study, a prototype platform, combining focused ion beam (FIB) and atomic force microscopy (AFM), was developed for 3D mapping of mechanical modulus and chemical signatures of interior regions of large biological structures with nanometer resolution. FIB milling provided high precision surface preparation and sample transfer to enable AFM probing of the sample interior. The prototype platform was also applied to image and investigate the fundamental mechanics of the rat face whiskers, a high-acuity sensor used to gain detailed information about the surrounding environment. Grazing angle FIB milling was first applied to expose the interior cross section of the rat whisker sample to AFM probing, followed by a ‘‘lift-out’’ method to retrieve and position the target sample for further analysis. To the best of the author’s knowledge, this is the first study to provide 3D insights into the interior of the rat whisker and a multilayer mechanical model by employing “FIB lift-out” technique for the preparation of AFM sample from biological materials. Although only applied to the rat whisker, the characterisation methodologies and protocols developed in this study could be extended to studies of a wide range of biomechanical systems in the future.
In the second part of this PhD research, by incorporating an ultrathin metallic coating on needle-shaped specimens prepared by FIB, a novel approach to analyse low conductive materials including biological cells with conventional pulsed-voltage atom probe tomography (APT) was proposed. Finite element electrostatic simulations of coated atom probe specimens were conducted, which showed remarkable improvements in conductivity and subsequent field evaporation of the biological samples with a nanoscale metallic coating of around 10 nm. After studying and comparing chemical deposition techniques and physical deposition techniques for APT specimen coating, a physical deposition coating technique, namely sputter coating, was chosen for ultra-thin nanoscale conductive coating of APT specimens. Using the design of experiments (DOE) technique, an experimental investigation was performed to study sputter coating of needle specimens with end tip radii less than 100 nm. The optimal coating strategy was applied for the coating of APT specimens prepared by the FIB lift-out technique, including resin embedded Au nanoparticles (NPs) as the control sample, and fixed dried bacterial cells. The results show that the optimal coating strategy allowed pulsed-voltage APT analysis and 3D imaging of low conductivity materials.
The optimal coating strategy was also applied to 3D near-atomic chemical mapping of susceptible and antibiotic resistant bacterial cells. Antibiotic resistance presents a significant global medical challenge, and the differences between the chemical composition and structure of susceptible and resistant strains are considered carrying the fundamental information of the underlying resistance mechanisms. Therefore, the proposed method of biological materials analysis with conventional pulsed-voltage APT was applied to acquire mass spectra and to reconstruct the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale from cell envelope and intracellular domains of polymyxin-susceptible and resistant strains of A. baumannii after treatment with polymyxin B. The results obtained from a comparison of data acquired from cell envelope and intracellular domains of susceptible and antibiotic resistant A. baumannii bacterial cells shed light on the compositional changes involved in the development of resistant mechanism. It is expected that the experimental approach demonstrated in the second part of this study will serve as a tool to investigate the architecture and chemistry of biological cells at the atomic-scale resolution in the future.
(1) Methods and protocols to explore the biological systems of size micrometre scale with nanometer resolution, including the capabilities to acquire mechanical and chemical information from interior regions of large biological structures.
(2) 3D nanoscale chemical mapping of biological materials, particularly biological cells.
The 3D data are essential to understanding the biological and cellular structure, while at the same time nanoscale resolution is necessary to follow the thinnest biological and cellular processes and to identify building blocks of biological and cellular structures such as RNA, DNA, and associated minute proteins. Thus, the principal purpose of this PhD research is divided into two parts: (1) to devise a prototype platform to investigate mechanical and chemical properties of interior regions of large biomechanical systems at nanometer scale, and (2) to develop an optimal strategy based on the advanced nanofabrication techniques for the applications of novel microscopy methods on 3D near-atomic chemical composition and structural characterization of biological materials, particularly biological cells.
In the first part of this PhD study, a prototype platform, combining focused ion beam (FIB) and atomic force microscopy (AFM), was developed for 3D mapping of mechanical modulus and chemical signatures of interior regions of large biological structures with nanometer resolution. FIB milling provided high precision surface preparation and sample transfer to enable AFM probing of the sample interior. The prototype platform was also applied to image and investigate the fundamental mechanics of the rat face whiskers, a high-acuity sensor used to gain detailed information about the surrounding environment. Grazing angle FIB milling was first applied to expose the interior cross section of the rat whisker sample to AFM probing, followed by a ‘‘lift-out’’ method to retrieve and position the target sample for further analysis. To the best of the author’s knowledge, this is the first study to provide 3D insights into the interior of the rat whisker and a multilayer mechanical model by employing “FIB lift-out” technique for the preparation of AFM sample from biological materials. Although only applied to the rat whisker, the characterisation methodologies and protocols developed in this study could be extended to studies of a wide range of biomechanical systems in the future.
In the second part of this PhD research, by incorporating an ultrathin metallic coating on needle-shaped specimens prepared by FIB, a novel approach to analyse low conductive materials including biological cells with conventional pulsed-voltage atom probe tomography (APT) was proposed. Finite element electrostatic simulations of coated atom probe specimens were conducted, which showed remarkable improvements in conductivity and subsequent field evaporation of the biological samples with a nanoscale metallic coating of around 10 nm. After studying and comparing chemical deposition techniques and physical deposition techniques for APT specimen coating, a physical deposition coating technique, namely sputter coating, was chosen for ultra-thin nanoscale conductive coating of APT specimens. Using the design of experiments (DOE) technique, an experimental investigation was performed to study sputter coating of needle specimens with end tip radii less than 100 nm. The optimal coating strategy was applied for the coating of APT specimens prepared by the FIB lift-out technique, including resin embedded Au nanoparticles (NPs) as the control sample, and fixed dried bacterial cells. The results show that the optimal coating strategy allowed pulsed-voltage APT analysis and 3D imaging of low conductivity materials.
The optimal coating strategy was also applied to 3D near-atomic chemical mapping of susceptible and antibiotic resistant bacterial cells. Antibiotic resistance presents a significant global medical challenge, and the differences between the chemical composition and structure of susceptible and resistant strains are considered carrying the fundamental information of the underlying resistance mechanisms. Therefore, the proposed method of biological materials analysis with conventional pulsed-voltage APT was applied to acquire mass spectra and to reconstruct the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale from cell envelope and intracellular domains of polymyxin-susceptible and resistant strains of A. baumannii after treatment with polymyxin B. The results obtained from a comparison of data acquired from cell envelope and intracellular domains of susceptible and antibiotic resistant A. baumannii bacterial cells shed light on the compositional changes involved in the development of resistant mechanism. It is expected that the experimental approach demonstrated in the second part of this study will serve as a tool to investigate the architecture and chemistry of biological cells at the atomic-scale resolution in the future.
