1,721,093 research outputs found
Real-time microdamage and strain detection during micromechanical testing of single trabeculae
No full textFor the assessment of local deformations, we recently combined mechanical testing of trabecular bone with high-speed photography. In a previous study on cuboids of human vertebral trabecular bone we found strained trabeculae to whiten, and scanning electron microscopy showed excessive microdamage in whitened zones. In the presented study we tested single trabeculae from bovine femur and tibia in a three-point-bending geometry. Whitening was detected in the form of an ellipsoid zone on the tension side on tested trabeculae. Upon crack formation the whitening fades and a whitened zone following the propagating crack tip can be seen In addition to whitening/microdamage assessment we use ink marks applied to the samples and a modified digital image correlation to obtain the local strains involved in whitening and formation. In the presented loading case the tensile strain along the long axis of the trabecula qualitatively correlates best with the whitening zones seen. Whitening and thus microdamage initiates around a local tensile strain of 3%, whereas crack initiation occurs at strains around 13%. Our results allow for the first time to correlate microdamage and local strains directly, which is direly needed for the development of realistic damage models used in finite element analyses
On recent developments for high-speed atomic force microscopy
No full textThe atomic force microscope (AFM) is limited in imaging speed by the bandwidth and dynamic behavior of the actuators and mechanical parts. For high-speed imaging all AFM components have to be optimized in performance. Here, we present improvements of the force sensor, the scanner, the controller, and the data acquisition system. By combining all these improvements, the next generation AFMs will enable imaging speeds more than two orders of magnitude faster than current commercial AFM systems
Advances in High-Speed Atomic Force Microscopy
Get PDFHigh-speed atomic force microscopy (HS-AFM) is a scanning probe technique capable of recording processes at the nanometre scale in real time. By sequentially increasing the speed of individual microscope components, images of surfaces can be recorded at up to several images per second. We present a HS-AFM platform composed of custom¿built measurement head, controller and software, scanners and amplifiers that is shared with the community in an open¿hardware fashion.
A new scanner design combined with an advanced control system is shown. The simple addition of a secondary actuator to widely available tube scanners increases the scan speed by over an order of magnitude while allowing for a 130 ¿m × 130 ¿m wide field of view, which is not possible with traditional high¿speed scanner designs. Controllers beyond standard proportional-integral controllers are capable of significantly increasing imaging speed by anticipating resonances. Such filters are cumbersome to design with conventional methods. It is shown how convex optimization can be used to design optimal controllers with guaranteed stability for atomic force microscopy in an automated fashion.
By integrating two lasers into the small spot¿size optics of an AFM readout head we are able to use the first laser for detecting the deflection of the smallest, and thus fastest currently available high¿speed cantilevers, while using the second for photo¿thermal actuation. Using this instrument, we demonstrate multi¿frequency atomic force microscopy (MF-AFM) at previously not accessible frequencies of more than 20 MHz.
By employing the driving laser not for resonant excitation as is usual in dynamic AFM, a new imaging mode, photothermal off-resonance tapping (PORT) is presented. By repeatedly thermally bending the cantilever below it¿s resonant frequency, the surface is probed at a rapid rate. The resulting force is extracted from the deflection of the cantilever in time¿ domain at real time and used for feedback and image generation. The dynamic and static force contributions in both PORT and state of the art high-speed amplitude modulation atomic force microscopy (AM-AFM) are measured and analyzed in detail. It is shown that by decoupling the driving frequency from the resonant frequency the dynamic tip¿sample impact forces can be drastically reduced when compared to resonance based AFM modes.
SAS-6 is a centriolar scaffolding protein with a crucial role in the duplication of centrioles, which are the main microtubule organizing organelle of eukaryotic cells. Defects in centriole duplication are associated with cancer and microencephaly. To understand these defects, is therefore important to understand the kinetics of SAS-6. In¿vitro, SAS-6 polymerizes into rings of between eight and ten monomers. Using the new PORT mode we are able to study the dynamic assembly of SAS-6. It is shown how SAS-6 rings can not only assemble by canonical one-by-one addition, but can form as a fusion of larger, already assembled fragments.
Finally, it is shown how PORT can be used to observe fast processes of and on living cells. The adhesion and detachment of thrombocyte cells is studied. Membrane disruptive effects are shown on gram¿negative as well as gram¿positive bacteria.LBN
A new versatile hybrid MEMS technology for high sensitivity, fluid proof sensing applications.
No full textThe field of micro electromechanical systems (MEMS) evolved from the microelectronic industry and the technologies developed to fabricate integrated circuits. As a result, MEMS are commonly fabricated on silicon wafers. The development of MEMS has been driven by three main merits: miniaturisation, microelectronics integration, and parallel fabrication with high precision. Many operational properties scale well with smaller sizes. In addition, integrated electronic circuitry allows embedding MEMS with computing or networking capabilities, while parallel manufacturing enables the fabrication of many identical devices on a single wafer, reducing the unit cost. The main MEMS application is transducers which transform signals from one form of energy to another, and can be used for perception (sensors) or to produce actions (actuators). Silicon and other microelectronic materials like metals have enabled very high-performance MEMS transducers because these materials can be used for a range of very effective actuating and sensing principles.
More recently, polymers have started to be used in MEMS because of their unique electrical, physical and chemical properties which include biocompatibility, viscoelasticity and mechanical shock tolerance. They are also much softer than silicon or metals, and they can be processed using many techniques that allow unique low-cost, batch-style fabrication and packaging. However, polymers have relatively low glass-transition and melting temperatures. As a result, the advantages of polymers cannot easily be combined with high-performance actuating and sensing elements as these are based on silicon technology and require high-temperature fabrication steps. Here, a hybrid-MEMS fabrication process is used to address this issue. The developed devices are based on a trilayer structure, where a thick polymer core is sandwiched between two hard thin films, while electronic layers are embedded within in a fluid-compatible way.
The objective of this thesis is to turn hybrid-MEMS into a benchmark MEMS technology. This involves many different aspects. First, sensing and actuation elements are integrated into trilayer devices, and their performance is analysed. Then, some more unique possibilities offered by hybrid-MEMS are exploited. A way to fabricate electronics on multiple layers is developed, which allows different electronic features to be integrated in parallel. In addition, three-dimensional bulk features fabricated at several levels are demonstrated. This is possible because the hybrid-MEMS process is based on the bonding of multiple wafers.
All these new fabrication features are demonstrated in atomic force microscopy (AFM) applications. Self-actuated trilayer AFM cantilevers with sharp silicon tips are used to boost imaging speeds in the off-resonance tapping (ORT) mode, thanks to an order of magnitude faster surface probing rates. Furthermore, fully integrated ORT imaging is demonstrated with self-actuated piezoresistive cantilevers. Next, trilayer cantilevers with shielded conductive tips are introduced for electrical AFM modes. Finally, ongoing research projects are touched upon, including approaches to fabricate hybrid-MEMS with single crystal silicon piezoresistors for improved sensitivity, and an analysis of reproducibility of fabricated trilayer cantilevers.LBN
A new technique for imaging mineralized fibrils on bovine bone fracture surfaces by atomic force microscopy
No full textA novel microfabrication platform for hybrid multilayer MEMS
No full textIn Atomic force microscopy (AFM), the tip-sample interaction force can be measured
through two primary detection techniques: optical beam detection (OBD) and electrical (self-sensing)
readout. Compared to the optical method, the convenience of the self-sensing
readout AFM measurements comes at the cost of higher force noise. In the self-sensing
method, there is a trade-off between reducing the force noise and maintaining the cantilever
characteristics (e.g. resonance frequency, spring constant, quality factor, and planar
dimension) within the practical limits. The core of my research was the development of
hybrid multilayer self-sensing cantilevers with up to one order-of-magnitude better force
sensitivity than state-of-the-art silicon self-sensing cantilevers. Thanks to a material
engineering approach combined with non-standard fabrication methods, the developed
cantilevers are designed such that a polymer core is sandwiched between two hard thin films.
The multilayer self-sensing cantilevers are designed to be thick and soft, thus combining
increased deflection sensitivity with low spring constants, and hence increasing the force
sensitivity.
The high force sensitivity of the hybrid multilayer cantilevers is accompanied by a high
detection bandwidth in AC modes. This originates from having a viscoelastic material as the
main structural layer, which causes low quality factor and hence high tracking bandwidth. In
terms of the imaging speed, the multilayer cantilevers show four times faster response
compared to their silicon counterparts. In addition, the hermetically sealed self-sensing
multilayer cantilevers can be deployed for various scanning probe microscopy (SPM)
applications in liquid as well as in air and vacuum with additional coatings.
For even further increase of the deflection sensitivity, newly developed high-gauge factor
strain sensors can be incorporated to the multilayer cantilevers governed by their adaptable
process flow. As a proof of concept, I show that atomically thin MoS2 piezoresistors can be
incorporated into SU8 cantilevers. However, the MoS2 piezoresistors have very high resistance, which has an adverse effect on the force noise of the cantilevers. One common strategy to alleviate this high resistance is doping the MoS2 piezoresistors. In this work, I show
that SU8 can act as a structural cantilever layer as well as an n-type doping source and an
encapsulation solution for the MoS2 piezoresistors.
In addition to the force resolution and the tracking ability, the quality and the
repeatability of any AFM image is also correlated with the cantilever tip shape (sharpness) and
durability. SU8 cantilevers have shown very good tracking ability but polymers are subjected
to high wear-rate as a tip material. In the scope of my research, I have also developed
fabrication recipes to integrate sharp, low wear-rate, silicon nitride tips into the pure SU8
cantilevers as well as the polymer-core multilayer cantilevers.
Furthermore, to extend the ease of use and versatility of AFM, a closed-loop scanner
based on a sidewall piezoresistive displacement sensor is presented. Such a closed loop scheme
compensates the piezotube scanner nonlinearities, namely hysteresis and creep. This closed-loop
system reshapes the piezotube drive signal through our developed FPGA-based
Proportional-Integral (PI) controller.LBN
Time-Resolved Scanning Ion Conductance Microscopy and Single-Molecule Spectroscopy
No full textThe evolution of the 3D morphology is at the center of many relevant biological processes ranging from cellular differentiation to cancer invasion and metastasis. Microscopy techniques, such as electron microscopy (EM), super-resolution (SR) optical microscopy, and atomic force microscopy (AFM), have been applied to image the structure of cells in great detail. The major challenge is to obtain 3D information at nanometer resolution without affecting the viability of the cells and avoiding interference with the process.
The thesis presents the development of a time-resolved scanning ion conductance microscope (SICM), from concept to prototype, capable of resolving spatiotemporal biological processes with unprecedented resolution and imaging speed. By integrating advances in nanopositioning, controls theory, microelectronics, and nanopore fabrication, the time-resolved SICM system enabled sub-5 nm resolution, performed high-speed imaging of 0.5 s per pixel, and allowed large imaging volumes. Moreover, the stability of the system enabled performing live cell imaging over 48 h without perturbations. We applied time-resolved SICM to dynamic processes on cell membranes that included: Structural evolution of circular dorsal ruffles (CDRs), shedding light on their mechanisms of formation; Morphological changes in human melanoma cells upon treatment with a drug known to reduce resistance to immunotherapy; And mechanisms of bacteria-host infection in the human cell membrane.
Furthermore, we combined time-resolved SICM with super-resolution fluorescence optical fluctuation microscopy (SOFI). By optimizing the SOFI computational approach and fluorophore's properties, high-speed correlative 3D imaging with mitigated phototoxic effects was achieved. The complementary capabilities of time-resolved SICM and SOFI provided comprehensive information on cell membrane morphology and cytoskeleton protein architecture, offering sub-diffraction resolution in living cells. This combined method holds promise as a routine tool for studying membrane processes.
In addition to imaging, time-resolved SICM was successfully adapted for single-molecule spectroscopy using nanopores, creating a new technique called scanning ion conductance spectroscopy (SICS). SICS overcame limitations in nanopore technology by controlling the nanopores' position and the translocation speed of individual molecules. The ability to control the speed and average multiple readings of the same molecule has resulted in two orders of magnitude increase in signal-to-noise ratio compared to conventional free translocation. In our glass nanopore experiments, we detected a 3.4 angstroms single nucleotide missing in a long strand of dsDNA. Moreover, we utilized SICS to successfully identify and analyze intricate topological features within complex DNA structures, including DNA-dCas9 complexes, hairpins, molecular rulers, and dsDNA gaps. The increased detection capability with SICS has the potential to be transferable to other solid-state and biological nanopore methods, significantly improving diagnostic and sequencing applications.LBN
Advances in self-sensing techniques for atomic force microscopy
Get PDFAtomic force microscope (AFM) is a tool that allows micro and nano scale imaging of samples ranging from solid state physics to biology. AFM uses mechanical forces to sense the sample and recreate a topography image with high spatial resolution. The biggest disadvantage of the standard AFMs is their scanning speed, as it typically takes up to several tens of minutes to capture an image. A lot of research was conducted to increase AFM scanning speed, which resulted in the development of high-speed AFMs (HS-AFMs), that can obtain an image in matter of seconds. Such increase in scanning speed enabled the study of various processes, ranging from functional mechanisms of proteins to cellular biology dynamics. Increasing the speed further, towards several tens of images per second would highly benefit many applications, from both material and life sciences. The imaging speed of an AFM is limited by the speed of its components. While scanners and electronic systems are constantly being improved, there exists a certain hold-up in the development of cantilevers and deflection sensing techniques. The mechanical bandwidth of the cantilever can be increased by decreasing its size. While it is possible to fabricate sub-micron sized cantilevers it becomes very challenging to sense their deflection. Standard AFMs rely on the optical beam deflection (OBD) readout, which can sense cantilevers down to 2 µm in width. Novel sensing techniques are needed to increase AFM imaging speed further. Strain-sensing techniques are particularly interesting as they offer many advantages over OBD readout, like the ability to sense sub-micron sized cantilevers. We investigated nanogranular tunneling resistors (NTRs) as strain-sensors for cantilever deflection sensing. With NTR ability to be deposited on various substrates and in arbitrary geometries, with lateral dimensions down to tens of nm and having reasonably high gauge factors, they are an interesting candidate for cantilever deflection sensing. We applied NTRs in AFM imaging for the first time, showing that their sensitivity is well suited for imaging of both solid state and biological samples. We also demonstrated that NTRs can be used for sensing of 500 nm wide cantilevers. We performed a study of doped Si piezoresistive strain sensors and of an unexploited potential which can be reached with the miniaturization of the cantilever dimensions. We demonstrated both theoretically and experimentally that by decreasing the size of the piezoresistive cantilevers, one can reach the AFM imaging noise performance equal or better than the noise performance of the OBD readout. We showed that piezoresistive cantilevers are very well suited for nm and Å scale imaging of both solid state and biological samples in air. In addition, we performed a research on an advancement of the AFM feedback controller. Most AFMs use digital signal processor (DSP) based feedback controllers. Digital implementation of the controller has some disadvantages, as it necessitates data converters which introduce additional delays in the feedback loop. We developed a fast digitally controlled analog proportional-integral-derivative (PID) controller. We successfully used this PID controller in AFM imaging, realizing several hundreds of Hz line rates. While the analog implementation of the controller provided large amplification and frequency bandwidth, digital control provided precise control of the system and reproducibility of parameter values.LBN
Advancements in Nanomechanical Characterization and Biomolecular Imaging with Atomic force Microscopy
No full textAtomic force microscopy (AFM) is a widely used imaging tool for obtaining a variety of information for a range of samples. Although it was initially intended to serve as a method of observing very flat solid surfaces, its use expanded into several other fields, such as high-speed biomolecular imaging, mechanical property measurement, and sample disruption. As a result, AFM continues to be an indispensable tool in research, contributing significantly to advancements in nanotechnology, biophysics, materials science, and numerous other interdisciplinary domains.
In this work, we apply a specific mode of high-speed AFM (HS-AFM) imaging called photothermal off-resonance tapping mode (PORT), which allows us to directly control the forces exerted on the sample. We use this method to image 2D assembly of DNA 3-point stars (3PS) to investigate the impact of structural flexibility and binding strength in the growth of supramolecular networks.
We then use a variation of slow off-resonance imaging called force volume to obtain the mechanical properties of biological membranes for organs-on-a-chip. We compare the utility and reliability of AFM to the bulge test assessment for known samples as well as a membrane aimed at mimicking the extracellular matrix (ECM) scaffold of in vivo barriers of lung tissue.
In the penultimate chapter, we demonstrate promising preliminary data on imaging clathrin mediated endocytosis on unroofed cells with AFM, and the effects of using cholesterol depletion to modify the biological process.
Finally, we discuss the contributions and remaining challenges related to imaging dynamic bioprocesses in vitro and in-vivo, particularly with the use of PORT. Through further development of individual AFM components, such as the cantilever, scanner, controller, and software, and combining them with fluorescent microscopy, we hope to obtain valuable information on self-assembling biosamples that would not be possible with other imaging methods.LBN
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