12 research outputs found
Atomic force microscopy-based mechanobiology
Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version
Mechanical oscillators probing cell mass and mechanics
The use of oscillators in science and technology has proven to be very impactful due to established mathematical concepts that laid the foundation for high accuracy measurements. In this thesis, I present how we used photo-thermally actuated mechanical oscillators to measure the mass and frequency-dependent mechanical properties of single, living cells with high accuracy and temporal resolution. We found that virus-infected cells stop cell growth, as well as that cell mass fluctuates within seconds by about 20 picograms which we could link to cellular water transport. Moreover, we could demonstrate the power-law behaviour of rounded cells up to a frequency of 40 kHz
pyIMD: Automated analysis of inertial mass measurements of single cells
The total mass of single cells can be accurately monitored in real time under physiological conditions with our recently developed picobalance. It is a powerful tool to investigate crucial processes in biophysics, cell biology or medicine, such as cell growth or hydration dynamics. However, processing of the raw data can be challenging, as computation is needed to extract the mass and long-term measurements can generate large amounts of data. Here, we introduce the software package pyIMD that automates raw data processing, particularly when investigating non-migrating cells. pyIMD is implemented in Python and can be used as a command line tool or as a stand-alone version including a graphical user interface
Hydrodynamic function and spring constant calibration of FluidFM micropipette cantilevers
Fluidic force microscopy (FluidFM) fuses the force sensitivity of atomic force microscopy with the manipulation
capabilities of microfluidics by using microfabricated cantilevers with embedded fluidic channels. This innovation
initiated new research and development directions in biology, biophysics, and material science. To acquire reliable and
reproducible data, the calibration of the force sensor is crucial. Importantly, the hollow FluidFM cantilevers contain a
row of parallel pillars inside a rectangular beam. The precise spring constant calibration of the internally structured
cantilever is far from trivial, and existing methods generally assume simplifications that are not applicable to these
special types of cantilevers. In addition, the Sader method, which is currently implemented by the FluidFM community,
relies on the precise measurement of the quality factor, which renders the calibration of the spring constant sensitive
to noise. In this study, the hydrodynamic function of these special types of hollow cantilevers was experimentally
determined with different instruments. Based on the hydrodynamic function, a novel spring constant calibration
method was adapted, which relied only on the two resonance frequencies of the cantilever, measured in air and in a
liquid. Based on these results, our proposed method can be successfully used for the reliable, noise-free calibration of
hollow FluidFM cantilevers
Entropic barrier of water permeation through single-file channels
Facilitated water permeation through narrow biological channels is fundamental for all forms of life. Despite its significance in health and disease as well as for biotechnological applications, the energetics of water permeation are still elusive. Gibbs free energy of activation is composed of an enthalpic and an entropic component. Whereas the enthalpic contribution is readily accessible via temperature dependent water permeability measurements, estimation of the entropic contribution requires information on the temperature dependence of the rate of water permeation. Here, we estimate, by means of accurate activation energy measurements of water permeation through Aquaporin-1 and by determining the accurate single channel permeability, the entropic barrier of water permeation through a narrow biological channel. Thereby the calculated value for ⳠS⥠= 2.01 ± 0.82 J/(mol·K) links the activation energy of 3.75 ± 0.16 kcal/mol with its efficient water conduction rate of ~1010 water molecules/second. This is a first step in understanding the energetic contributions in various biological and artificial channels exhibiting vastly different pore geometries.ISSN:2399-366
Tailoring the Sensitivity of Microcantilevers To Monitor the Mass of Single Adherent Living Cells
Microcantilevers are widely employed as mass sensors for biological samples, from single molecules to single cells. However, the accurate mass quantification of living adherent cells is impaired by the microcantileverâs mass sensitivity and cell migration, both of which can lead to detect masses mismatching by â«50%. Here, we design photothermally actuated microcantilevers to optimize the accuracy of cell mass measurements. By reducing the inertial mass of the microcantilever using a focused ion beam, we considerably increase its mass sensitivity, which is validated by finite element analysis and experimentally by gelatin microbeads. The improved microcantilevers allow us to instantly monitor at much improved accuracy the mass of both living HeLa cells and mouse fibroblasts adhering to different substrates. Finally, we show that the improved cantilever design favorably restricts cell migration and thus reduces the large measurement errors associated with this effect.ISSN:1530-6984ISSN:1530-699
Rheology of rounded mammalian cells over continuous high-frequencies
Understanding the viscoelastic properties of living cells and their relation to cell state and morphology remains challenging. Low-frequency mechanical perturbations have contributed considerably to the understanding, yet higher frequencies promise to elucidate the link between cellular and molecular properties, such as polymer relaxation and monomer reaction kinetics. Here, we introduce an assay, that uses an actuated microcantilever to confine a single, rounded cell on a second microcantilever, which measures the cell mechanical response across a continuous frequency range â 1â40âkHz. Cell mass measurements and optical microscopy are co-implemented. The fast, high-frequency measurements are applied to rheologically monitor cellular stiffening. We find that the rheology of rounded HeLa cells obeys a cytoskeleton-dependent power-law, similar to spread cells. Cell size and viscoelasticity are uncorrelated, which contrasts an assumption based on the Laplace law. Together with the presented theory of mechanical de-embedding, our assay is generally applicable to other rheological experiments.ISSN:2041-172
Monitoring the mass, eigenfrequency, and quality factor of mammalian cells
Abstract The regulation of mass is essential for the development and homeostasis of cells and multicellular organisms. However, cell mass is also tightly linked to cell mechanical properties, which depend on the time scales at which they are measured and change drastically at the cellular eigenfrequency. So far, it has not been possible to determine cell mass and eigenfrequency together. Here, we introduce microcantilevers oscillating in the Ă
ngström range to monitor both fundamental physical properties of the cell. If the oscillation frequency is far below the cellular eigenfrequency, all cell compartments follow the cantilever motion, and the cell mass measurements are accurate. Yet, if the oscillating frequency approaches or lies above the cellular eigenfrequency, the mechanical response of the cell changes, and not all cellular components can follow the cantilever motions in phase. This energy loss caused by mechanical damping within the cell is described by the quality factor. We use these observations to examine living cells across externally applied mechanical frequency ranges and to measure their total mass, eigenfrequency, and quality factor. The three parameters open the door to better understand the mechanobiology of the cell and stimulate biotechnological and medical innovations
High-resolution mass measurements of single budding yeast reveal linear growth segments
The regulation of cell growth has fundamental physiological, biotechnological and medical implications. However, methods that can continuously monitor individual cells at sufficient mass and time resolution hardly exist. Particularly, detecting the mass of individual microbial cells, which are much smaller than mammalian cells, remains challenging. Here, we modify a previously described cell balance (âpicobalanceâ) to monitor the proliferation of single cells of the budding yeast, Saccharomyces cerevisiae, under culture conditions in real time. Combined with optical microscopy to monitor the yeast morphology and cell cycle phase, the picobalance approaches a total mass resolution of 0.45 pg. Our results show that single budding yeast cells (S/G2/M phase) increase total mass in multiple linear segments sequentially, switching their growth rates. The growth rates weakly correlate with the cell mass of the growth segments, and the duration of each growth segment correlates negatively with cell mass. We envision that our technology will be useful for direct, accurate monitoring of the growth of single cells throughout their cycle.ISSN:2041-172
Atomic force microscopy-based mechanobiology
Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer Reviewe