Paul K. Hansma - US grants

This research focuses on finding the fundamental limits on the performance of Atomic Force Microscopes (AFMs), and then building AFMs that come closer to those fundamental limits. Development of AFMs for small cantilevers that can be used by scientists and engineers across many disciplines is a primary goal of this research. Small cantilevers have higher resonant frequencies at a given spring constant, thus the thermal noise is spread out over a larger frequency range giving less noise per unit bandwidth. Since small cantilevers have lower viscous drag in solution less dissipation and smaller fluctuations are observed. This allows faster and gentler imaging of soft materials, such as biological samples. A strong motivation in this research and development is the long-term goal of making AFMs that can be useful on a wide range of materials, such as enzymes and other proteins for biomaterials processing. Therefore, small cantilevers and small cantilever AFMs could be an important part of the future of scanning probe microscopy, which will be of fundamental and practical interest to materials research. Graduate students involved in the project receive training in fundamental experimental techniques with cutting edge technology. This training will prepare them for a range of careers in academe, industry or government.
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This research focuses on finding the fundamental limits on the performance of Atomic Force Microscopes (AFMs), and then building AFMs that come closer to those fundamental limits. Based on previous research, we know that small cantilevers will be essential for this goal. Thus, we will build AFMs for small cantilevers with goals not only of coming closer to the fundamental limits, but also of making generally useful microscopes for a wide range of applications in materials research and beyond. On application that seems especially promising is single molecule mechanics of enzymes that nature uses for materials synthesis. By analyzing single molecule mechanics of enzymes and other proteins, scientists and engineers will be able to develop materials that are environmentally friendly, easy to manufacture (e.g. under ambient conditions), and cost effective. Such materials would span medical and textile industries. Graduate students involved in the project receive training in fundamental experimental techniques with cutting edge technology. This training will prepare them for a range of careers in colleges, industry or government.

This is a proposal to develop the DNA sequencing technology using the atomic force microscope (AFM). AFM can image surfaces at submolecular resolution by scanning a sharp tip over the surface, and under favorable conditions, and has resolved individual nucleotides of DNA. In this project, feasibility studies will be carried out to determine if, in fact, reliable methods could be developed for AFM to actually sequence DNA molecules. If successful, the new method could allow DNA sequencing at least 100 times faster than the currently available technology.

Biomineralized materials such as molluscan shells are complex lamellar aggregates of inorganic single crystals and biopolymers that form at low temperature with exceptional strength and crystalline ordering. The biosynthesis process gives and incredible regularity to 1) inorganic and protein structure at nanoscale (atomic) dimensions, and 2) the initiation and termination of growth at mesoscale (is greater than approximately 0.1 microns) dimensions, thus generating the highly ordered microlaminate composite formation that gives the resulting biomaterial its strength and flexibility. This interdisciplinary research will investigate the fundamental chemical and stereochemical interactions which, at the molecular level control such biologically mediated mineral nucleation and growth at both the nano- and mesostructure level. Nucleation and growth of inorganic phases will be carried out from solutions at low temperatures and pressures on: i) inorganic and biological mineral templates; ii) synthetic organic templates with optically active groups and surface arrays of ionic sites similar to those found on biopolymeric templating surfaces in vivo; and iii) biogenic templating polypeptides. The proposed experiments will use a novel combination of synthesis and characterization techniques, including polypeptide sequencing and synthesis to form structure directing templates, in situ atomic force microscopy and grazing incidence X-ray diffraction. %%% Nature's use of templating involves material growth from solutions at low temperatures and pressures, a process that is currently only poorly understood and little used by materials scientists. Increasingly, it is becoming apparent that these new materials, which have been referred to as "chemically bonded ceramics", have exceptional flexural strength. The proposed activity of this research is expected to lead to the development of new methods for the low temperature synthesis of mechanically superior and/or highly oriented materials using nanostructure design and synthesis techniques. It is hoped that as more light is shed on templating mechanisms involved, templating kinetics will be improved and template-directed materials synthesis can be done on a faster time scale. While the research will initially be centered around the mineralization processes of molluscs, which specifically involves CaCO3 growth, all multicellular organisms appear to have evolved with chemically similar tools for carrying out genetic control over mineral nucleation and growth. We therefore expect that our experimentation will increase insight on related areas of biomineralization, including bone growth. By more fully realizing how mineralization is orchestrated in one particular species, the answers to more complex biomineralization problems will surely follow. Implications of our results for the medical field are foreseeable; bone nucleation growth is based on the same fundamental chemical and structural interactions as occurs in mollusc shells, except that the nucleating macromolecules are part of a bilipid membrane which encloses the mineralization space. Thus, the study of hydroxyapatite nucleation onto such lipid bilayer surfaces may follow. On the other hand, a greater understanding of soluble growth inhibitors can be useful for the prevention of undesirable crystal growth. Medical examples include dental plaque, organ stones, hardening of the arteries, and calcification of implanted heart valves. In industrial cooling systems, oil recovery systems, and municipal water supplies, undesirable water-formed scale deposits of CaCO3 and CaSO4 foul treatment facilities. New organic compounds may inhibit scale formation, be cost effective, and be useful over a larger range of temperatures, pH, and salinity.

The objective of this project is to build an instrument that will allow rapid evolution of sharp and special purpose tips for scanning probe microscopes. This new instrument integrates a scanning probe microscope with a scanning electron microscope and ion mill for preparing, imaging and using probe tips. Of special interest are processes involving decomposition of gases and liquid films to form hard tips, ferroelectric tips, and magnetic tips. The significance is that almost all scanning probe measurements, expecially with the atomic force microscope, are limited now by the tip. For the atomic force microscope, there is no instrument presently available that allows researchers to do rapid tip development. The system proposed here will allow super tips to be optimized rapidly, a benefit to many users of the atomic force microscope.

9317466 Hansma DNA can now be routinely and reproducibly imaged with the Atomic Force Microscope (AFM) in air and in a variety of fluids, including aqueous buffers. Building on this demonstrated capability, the following 5 research areas are proposed: (1) To image the effects of drug-binding on DNA conformation, using distamycin and other peptides that induce conformation-changes in AT-rich regions of DNA. (2) To increase the resolution of DNA-imaging by using new improved tips and imaging environments selected for minimizing both adhesion and repulsion. Tips will be fabricated with a new AFM-SEM (scanning electron microscope) combination that is currently being built. (3) To label DNA with a variety of reliably distinguishable labels for AFM-mapping of DNA at a resolution of several dozen nucleotides. (4) To improve the quality of imaging of single- stranded DNA. The ultimate goal of this technology is the high- speed sequencing of single DNA molecules. (5) To image processes involving DNA-protein interactions in real time, such as the cleavage of DNA with a restriction enzyme and the interaction of single-stranded and double-stranded DNA with RecA protein. %%% The Atomic Force Microscope (AFM) s a new microscope, invented in 1986, that can feel surfaces with enough sensitivity to map the size and shape of DNA and other biological molecules. For many applications, the AFM combines the convenience and versatility of optical microscopy with a resolution comparable to electron microscopy. Since this high resolution can be obtained even in aqueous solutions, the AFM can image molecules that are biologically active and has the capability of imaging these molecules in action. Atomic force microscopy of DNA has progressed rapidly in the last 2 years. New research can now build on this foundation. ***

9724254 Safinya Optical imaging instrumentation consisting of a laser scanning confocal microscope with real-time video-rate capability and a micromanipulation system will be acquired by the University of California at Santa Barbara. The instrument will support real- time studies of novel biomolecular materials, confined complex fluids, ceramics, alloys and composites. It will be housed within the Materials Research Laboratory at UCSB, where it will be accessible to all campus researchers. %%% Acquisition of this instrumentation will impact research and research training in an interdisciplinary program at UCSB in the general area of complex materials that involves about 30 graduate students, 15 post doctoral associates and 11 faculty. ***

DESCRIPTION (provided by applicant): Proteins have many structural and mechanical roles in the body from collagen in bones to titin in muscles and from integrin on the cell surface to ion channels within cell membranes. Recently, it has been shown that special Atomic Force Microscopes, "pullers," can be used to measure the mechanical properties of individual molecules by pulling on them. The force vs. distance curves from these pullers contain information about bonds within molecules and about bonds between molecules. This information could lead to a deeper understanding of pathologies associated with the structural and mechanical properties of proteins and give insight into possible remedies. There are, however, problems with the existing pullers that are available for general use. As examples: 1) the force noise in the pullers currently used for this work is significantly larger than it needs to be, obscuring subtle features due to weak bonds. 2) Existing pullers do not combine the Atomic Force Microscope's imaging abilities for selecting molecules together with accurate sensors to measure the distance of pulling. Other problems are detailed in the body of the proposal. Work is proposed here to improve the instruments that are available for general use by three stages of instrument development: (1) adding small cantilever capability for increased force resolution (a factor of 5) and increased speed (a factor of 30) while retaining accurate position measurement and imaging, (2) adding optical access for microscope objectives with high numerical aperture and (3) moving only the cantilever in both pulling and imaging to avoid high frequency shaking of delicate samples like cell cultures.

[unreadable] DESCRIPTION (provided by applicant): The Atomic Force Microscope (AFM) can image and manipulate biological samples under physiological conditions at nanometer resolution. Despite its very slow operation (sometimes over 30 minutes per image) and the special expertise required to overcome its complexity of use, the AFM has already facilitated significant biomedical discoveries including new insights into membrane proteins and the mechanical properties of structural proteins. The range of biological processes the AFM can image is, however, severely limited by the AFM's current low time resolution, which is due to fundamental limits inherent in the current generation of AFMs based on cantilevers with dimensions of order 100 microns. This new generation of user-friendly (2 hours' training), high speed (2 images/second) AFMs is proposed to be developed based on fundamental innovations in design, particularly by basing the new AFMs on cantilevers with dimensions of order 10 microns to improve time and force resolution for probing molecules and speed in imaging by an order of magnitude or more. The Specific Aims of the project are: i) to develop a high-speed AFM that can image at 512x512 resolution, at 2 images/second; 2) to develop a user-friendly AFM in which most of the processes in setting up the AFM and adjusting feedback parameters are automated; 3) to develop a new AFM that combines high-speed with automation. It is hoped and expected that these new AFMs can serve as prototypes to guide the development of future commercial AFMs, which can bring the advantages of high-speed and automation to the broader community of biomedical researchers. This new generation of AFMs will be fast and easy to use and have improved tune and force resolution. This will facilitate imaging of biological processes at the molecular level and thus have a broad enabling impact on biomedical research. The long term goal is to transition the AFM from a specialized research tool to a general use medical instrument. [unreadable] [unreadable] [unreadable]

The research objective of this Collaborative Research award is to develop capabilities for machining structural biological composites such as bone and nacre with minimal surface damage. These biological composites exhibit many levels of hierarchical structures from macroscopic to microscopic length scales, and novel structural mechanical properties. The research approach will utilize novel diagnostic instruments for studying deformation/fracture in biological composites and high-resolution optical techniques for analysis of deformation in machining. Modeling structured around finite element analysis and moving heat source theory will be used to predict the thermomechanical state of the composite surfaces created by cutting. Model predictions will be compared with the experimental observations. By integrating these results, recommendations will be developed for improved cutting procedures that will minimize surface damage in this class of structural bio-materials.

If successful, the broader impact of the results will be new cutting methodologies for the bio-inspired structural materials of the future, and improved procedures for surgery that minimize damage to bone and tissue. The results will be of value also in understanding deformation and cutting of complex bio-inspired (structural) materials of the future and soft matter, and for continued development of diagnostic instrumentation for biological composites. Complementing the research is an education and training program that includes development of a bone cutting test-bed for practitioners and interns, student internships, seminar series webcast, and a modest focus on fostering entrepreneurship in graduate study.

The research objective of this award is to create a strong technological base that will enable bio-inspired sensors and actuators to improve the safety, reliability, longevity, and functionality of civil and mechanical infrastructures; and to make a contribution to the process of intelligent renewal through the support of research and education focusing on interdisciplinary collaboration. The approach will focus on using bio-inspiration from both biomaterials and biological organisms to develop novel actuator systems - especially passive actuator systems with tunable parameters - to mitigate the damage from natural disasters such as earthquakes and wind. Novel actuator systems in an experimental shear beam model structure will be used to compare experimental results to theoretical results. This comparison will inform the development of reliable computational tools for modeling and optimizing the performance of passive actuator systems with tunable parameters as well as informing the design of optimized passive actuators.

If successful, this research has the potential to revitalize the field of structural protection with passive actuators. It will combine fundamental scientific and engineering principles with state-of-the-art technology, new paradigms and new prototypes inspired by nature's technology. This research will enhance interdisciplinary education with a commitment to outreach, recruitment, and retention of underrepresented minority and woman students. The PI and Co-PIs will provide outreach the High Schools and summer participation programs to get high school students involved in laboratory research. The project will support two graduate students in an interdisciplinary environment that will broaden their experience beyond what they would gain in a single discipline. The PI and Co-PIs will make every effort to recruit minority and woman students to work on the project both as graduate research assistants and as undergraduate researchers. The project will emphasize strong ties to California's MESA (Mathematics, Engineering, Science Achievement) Program, which presents great opportunities for involving educationally disadvantaged students in research.

DESCRIPTION (provided by applicant): A new tool is necessary to explore the nanoscale mechanisms that contribute to tissue mechanical properties. These nanoscale mechanisms are of key importance in understanding bone fragility, intervertebral disc degeneration, osteoarthritis, and other diseases. The Atomic Force Microscope, AFM, with its ability to both image and manipulate nanostructures, has been a powerful tool in this area. But it has a major limitation for studying tissue samples: most tissue samples are too rough to be imaged with AFM cantilevers. Here we propose to move beyond cantilevers to a novel Deep AFM probe that enables a vertical approach deep into the topography of tissue samples. The objectives of the proposed research are to build the first prototypes of a new generation of Atomic Force Microscopes, Deep AFMs, that will increase imaging range of AFMs by at least one order of magnitude, and then to use these Deep AFMs to explore tissue structures and nanomechanics with a goal of understanding the molecules and nanoscale processes involved in tissue degeneration at a sufficient level of detail to inform development of new therapies. The overall impact and relevance of the proposed work is broad due to the numerous potential applications, however, because of our current progress and work on bone diagnostics, we will focus primarily on the nanoscale fracture mechanics of bone. We propose three related aims that include both the development and characterization of this new class of AFMs as well as their application in clinically relevant bone tissue samples. Specific Aim 1 is to develop Deep AFM I for very large scale scanning and nanomechanics. It will enable imaging of crack propagation in bone submerged in buffer as well as spatially resolved force spectroscopy, nanomanipulation and indentation to measure local nanomechanical properties. Specific Aim 2 is to develop Deep AFM II for high resolution, large scale scanning and nanomechanics. The higher resolution of Deep AFM II will enable imaging the nanoscale origin of bone fracture cracks with resolution comparable to Scanning Electron Microscopy, but without ever removing the sample from buffer. Thus it will be possible to image nanoscale processes that occur at intermediate stages of the crack growth process. It will also be possible to perform spatially resolved force spectroscopy, nanomanipulation and indentation to measure local nanomechanical properties. Specific Aim 3 is to use Deep AFM to move forward in our understanding of the nanoscale mechanisms of bone fracture and ways to reduce bone fracture risk. With Deep AFM, we can continue to move toward a long term goal of clinically decreasing the component of bone fracture risk by understanding the molecules and nanoscale mechanisms that resist bone fracture. PUBLIC HEALTH RELEVANCE: Bone fragility, intervertebral disc degeneration, cancer, atherosclerosis, osteoarthritis, and tooth decay all involve changes in tissue mechanical properties at the nanoscale. The proposed Deep Atomic Force Microscope, Deep AFM, is designed to investigate these changes with the goal of understanding how to prevent and even reverse undesirable changes for tissues in general and bone in particular. This work will be synergistic with our ongoing collaboration with physicians on diagnosing bone fragility due to bone tissue degeneration in patients.

DESCRIPTION (provided by applicant): Osteoporosis is a major medical problem affecting over 44 million individuals in the US, and a substantial number of those individuals will experience a low impact fracture. To effectively assess fracture risk in this population, one must be able to directly measure bone quality parameters in a patient that reflects the overall mechanical competence of the bone tissue. No current method or instrument can safely and practically perform that task. The BioDent Reference Point Indenter has been the subject of laboratory research for over six years, and recent studies support its ability to measure critical bone properties in osteoporotic individuals. BioDent quantifies bone tissue microfractures through the use of a microindentation process, and there is a strong relationship between microfracture and general fracture incidence. The central hypotheses of this proposal are (HP 1) minimally invasive micro-level in situ bone quality measurements via Reference Point Indentation are robust indicators of organ-level bone fragility; (HP 2) Reference Point Indentation measurements made at the tibiae exhibit similar trends of fragility as the common fracture sites within the same donor; and (HP 3) Reference Point Indentation measurements can further discriminate bone fragility with higher fidelity and accuracy than BMD. The proposed study will rigorously test these hypothesis using the BioDent Reference Point Indentation system, mechanical testing, and DeXA to demonstrate that minimally invasive indentations can predict bone fragility to a greater degree of accuracy than DeXA. PUBLIC HEALTH RELEVANCE: There are over 44 million osteoporosis patients in the US. Currently, no diagnostic method can predict the high number of low impact fractures observed among those individuals. The proposed study is directed to an instrument that safely and directly measures the resistance of bone material to fracture.

Organisms in Nature have crafted tools and processes to perform tasks such as foraging, eating, hunting, and burrowing with high efficiency. This award supports fundamental research that will examine mechanisms of cutting processes used by various organisms in the natural world, so as to identify new concepts that can potentially be translated into the industrial manufacturing domain. Research results can potentially lead to a class of highly efficient manufacturing processes for cutting and deformation processing of soft matter, and impact pharmaceutical manufacturing and orthopedic surgery. Given the critical importance of surgical cutting ranging from soft tissue excision to more serious orthopaedic procedures, the research could also contribute to improving quality of life by accelerating post-surgery healing.

The research objectives are: (1) identification of some typical mechanisms employed by insects, rodents, and other small animals in cutting and indentation processes in the natural world, and compare and contrast these against conventional manufacturing processes with soft matter in terms of efficiency and material removal mechanics; (2) characterization of key mechanical properties of soft natural material systems, relevant to these select processes, which determine their cutting and indentation response; (3) obtain fundamental knowledge on manufacturing processes used in the natural world and explain their applicability to manufacturing. To achieve the first research objective, experimental studies will be conducted on cutting and indentation processes in nature. The tool systems will include animal teeth (canine and equine) and insect mandibles (leaf cutter ants and beetles) such as mouthparts used for biting, cutting, and crushing. Experiments will be performed in both a simulated natural environment in a glass container within the lab and through instrumented lab-scale model systems. Direct observations will be performed using high speed imaging and microscopy, coupled with quantitative analysis by image correlation techniques. To achieve the second objective, mechanical properties and surface characteristics of natural tools and work materials (e.g., leaves, wood, bone, and flesh) will be examined using Atomic Force Microscopy, nano-indentation, micromechanical testing, and electron microscopy. To achieve the third objective, numerical modeling will be used to analyze the mechanics of cutting and indentation processes in two select natural systems. This modeling will be validated by experiments and then used for benchmarking against industrial cutting processes such as comminution and punching. The modeling tools will be Finite Element Analysis and moving heat source theory.