Jorge Otero

Nanocharacterization of soft biological samples in shear mode with quartz tuning fork probes.

Quartz tuning forks are extremely good resonators and their use is growing in scanning probe microscopy. Nevertheless, only a few studies on soft biological samples have been reported using these probes. In this work, we present the methodology to develop and use these nanosensors to properly work with biological samples. The working principles, fabrication and experimental setup are presented. The results in the nanocharacterization of different samples in different ambients are presented by using different working modes: amplitude modulation with and without the use of a Phase-Locked Loop (PLL) and frequency modulation. Pseudomonas aeruginosa bacteria are imaged in nitrogen using amplitude modulation. Microcontact printed antibodies are imaged in buffer using amplitude modulation with a PLL. Finally, metastatic cells are imaged in air using frequency modulation.

Finite Element Analysis of Electrically Excited Quartz Tuning Fork Devices

Quartz Tuning Fork (QTF)-based Scanning Probe Microscopy (SPM) is an important field of research. A suitable model for the QTF is important to obtain quantitative measurements with these devices. Analytical models have the limitation of being based on the double cantilever configuration. In this paper, we present an electromechanical finite element model of the QTF electrically excited with two free prongs. The model goes beyond the state-of-the-art of numerical simulations currently found in the literature for this QTF configuration. We present the first numerical analysis of both the electrical and mechanical behavior of QTF devices. Experimental measurements obtained with 10 units of the same model of QTF validate the finite element model with a good agreement.

Bidirectional mechanobiology between cells and their local extracellular matrix probed by atomic force microscopy

There is growing recognition that the mechanical interactions between cells and their local extracellular matrix (ECM) are central regulators of tissue development, homeostasis, repair and disease progression. The unique ability of atomic force microscopy (AFM) to probe quantitatively mechanical properties and forces at the nanometer or micrometer scales in all kinds of biological samples has been instrumental in the recent advances in cell and tissue mechanics. In this review we illustrate how AFM has provided important insights on our current understanding of the mechanobiology of cells, ECM and cell-ECM bidirectional interactions, particularly in the context of soft acinar tissues like the mammary gland or pulmonary tissue. AFM measurements have revealed that intrinsic cell micromechanics is cell-type specific, and have underscored the prominent role of β1 integrin/FAK(Y397) signaling and the actomyosin cytoskeleton in the mechanoresponses of both parenchymal and stromal cells. Moreover AFM has unveiled that the micromechanics of the ECM obtained by tissue decellularization is unique for each anatomical compartment, which may support both its specific function and cell differentiation. AFM has also enabled identifying critical mechanoregulatory proteins involved in branching morphogenesis (MMP14) and acinar differentiation (α3β1 integrin), and has clarified the role of altered tissue mechanics and architecture in a variety of pathologic conditions. Critical technical issues of AFM mechanical measurements like tip geometry effects are also discussed.

Multitool Platform for Morphology and Nanomechanical Characterization of Biological Samples With Coordinated Self-Sensing Probes

Single-cell studies are extremely important in several fields of research in both molecular and cell biology. Current nanocharacterization techniques based on atomic force microscopy allow researchers to study cells and molecules in unprecedented detail. An important limitation of conventional equipment results from the use of a single probe to measure different parameters of the same sample. To avoid this, here we present a multitool platform based on coordinated self-sensing probes. A first tool, based on quartz tuning fork resonators, is used to image the surface. A second tool, based on a piezoresistive cantilever, is used to measure the nanomechanical properties of the sample. Specific instrumentation circuitry was developed to optimize imaging and force measurement with these probes. Different coordination strategies were studied and a solution was selected that coordinates the nanotools in the microworld and in the nanoworld. Finally, an experiment with biological samples was conducted: the tuning-fork-based tool measured the topography of Escherichia Coli bacterial membranes and the piezoresistive-cantilever-based tool measured their elastic properties.

Quartz tuning fork studies on the surface properties of Pseudomonas aeruginosa during early stages of biofilm formation

Scanning probe microscopy techniques are powerful tools for studying the nanoscale surface properties of biofilms, such as their morphology and mechanical behavior. Typically, these studies are conducted using atomic force microscopy probes, which are force nanosensors based on microfabricated cantilevers. In recent years, quartz tuning fork (QTF) probes have been used in morphological studies due to their better performance in certain experiments with respect to standard AFM probes. In the present work QTF probes were used to measure not only the morphology but also the nanomechanical properties of Pseudomonas aeruginosa during early stages of biofilm formation. Changes in bacterium size and the membrane spring constant were determined in biofilms grown for 20, 24 and 28 h on gold with and without glucose in the culture media. The results obtained using the standard AFM and QTF probes were compared. Both probes showed that the bacteria forming the biofilm increased in size over time, but that there was no dependence on the presence of glucose in the culture media. On the other hand, the spring constant increased over time and there was a clear difference between biofilms grown with and without glucose. This is the first time that QTF probes have been used to measure the nanomechanical properties of microbial cell surfaces and the results obtained highlight their potential for studying biological samples beyond topographic measurements.

Improving the lateral resolution of quartz tuning fork-based sensors in liquid by integrating commercial AFM tips into the fiber end

The use of quartz tuning fork sensors as probes for scanning probe microscopy is growing in popularity. Working in shear mode, some methods achieve a lateral resolution comparable with that obtained with standard cantilevered probes, but only in experiments conducted in air or vacuum. Here, we report a method to produce and use commercial AFM tips in electrically driven quartz tuning fork sensors operating in shear mode in a liquid environment. The process is based on attaching a standard AFM tip to the end of a fiber probe which has previously been sharpened. Only the end of the probe is immersed in the buffer solution during imaging. The lateral resolution achieved is about 6 times higher than that of the etched microfiber on its own.

Determination of the static spring constant of electrically-driven quartz tuning forks with two freely oscillating prongs.

Quartz tuning forks have become popular in nanotechnology applications, especially as sensors for scanning probe microscopy. The sensors spring constant and the oscillation amplitude are required parameters to evaluate the tip-sample forces; however, there is certain controversy within the research community as to how to arrive at a value for the static spring constant of the device when working in shear mode. Here, we present two different methods based on finite element simulations, to determine the value of the spring constant of the sensors: the amplitude and Cleveland methods. The results obtained using these methods are compared to those using the geometrical method, and show that the latter overestimates the spring constant of the device.

A Feedfordward Adaptive Controller to Reduce the Imaging Time of Large-Sized Biological Samples with a SPM-Based Multiprobe Station

The time required to image large samples is an important limiting factor in SPM-based systems. In multiprobe setups, especially when working with biological samples, this drawback can make impossible to conduct certain experiments. In this work, we present a feedfordward controller based on bang-bang and adaptive controls. The controls are based in the difference between the maximum speeds that can be used for imaging depending on the flatness of the sample zone. Topographic images of Escherichia coli bacteria samples were acquired using the implemented controllers. Results show that to go faster in the flat zones, rather than using a constant scanning speed for the whole image, speeds up the imaging process of large samples by up to a 4× factor.

Micropattern of antibodies imaged by shear force microscopy: comparison between classical and jumping modes.

Quartz tuning fork devices are increasingly being used as nanosensors in Scanning Probe Microscopy. They offer some benefits with respect to standard microfabricated cantilevers in certain experimental setups including the study of biomolecules under physiological conditions. In this work, we compare three different working modes for imaging micropatterned antibodies with quartz tuning fork sensors: apart from the classical amplitude and frequency modulation strategies, for first time the jumping mode is implemented using tuning forks. Our results show that the molecules suffer less degradation when working in the jumping mode, due to the reduction of the interaction forces.

Implementation of a Multirobot System using Self-sensing Probes for Cell and Molecular Biology Experimental Research

Abstract Atomic Force Microscopy (AFM) is a well known technique in cell and molecular biology research. AFM can measure morphological, nanomechanical and biochemical properties of living cells with unprecedented level of detail. But the technique still presents some limitations. The main one is the use of the same probe for all the tasks. To overcome this limitation, authors present in this work the implementation of a mutiprobe robotic station. The station has nanometric resolution positioning, submicron resolution referencing between different agents and high versatility for the integration of different nanotools: we present the integration of displacement nanosensors based on quartz tuning forks and force nanosensors based on piezoresistive cantilevers. Results from the test reported with these nanosensors in this work show an imaging resolution better than 2nm and a force resolution better than 5nN. Then, with the robotic station optimized, nanomechanical measurements of bacterial membranes are performed. The obtained results show a good agreement with measurements done with a commercial AFM and with the values reported in the literature.