9,239 research outputs found
Nanomechanical and topographical imaging of living cells by Atomic Force Microscopy with colloidal probes
Atomic Force Microscopy (AFM) has a great potential as a tool to characterize
mechanical and morphological properties of living cells; these properties have
been shown to correlate with cells' fate and patho-physiological state in view
of the development of novel early-diagnostic strategies. Although several
reports have described experimental and technical approaches for the
characterization of cell elasticity by means of AFM, a robust and commonly
accepted methodology is still lacking. Here we show that micrometric spherical
probes (also known as colloidal probes) are well suited for performing a
combined topographic and mechanical analysis of living cells, with spatial
resolution suitable for a complete and accurate mapping of cell morphological
and elastic properties, and superior reliability and accuracy in the mechanical
measurements with respect to conventional and widely used sharp AFM tips. We
address a number of issues concerning the nanomechanical analysis, including
the applicability of contact mechanical models and the impact of a constrained
contact geometry on the measured elastic modulus (the finite-thickness effect).
We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in
order to demonstrate the importance of the correction of the finite-thickness
effect and the change in cell elasticity induced by the action of a
cytoskeleton-targeting drug.Comment: 51 pages, 12 figures, 3 table
A practical review on the measurement tools for cellular adhesion force
Cell cell and cell matrix adhesions are fundamental in all multicellular
organisms. They play a key role in cellular growth, differentiation, pattern
formation and migration. Cell-cell adhesion is substantial in the immune
response, pathogen host interactions, and tumor development. The success of
tissue engineering and stem cell implantations strongly depends on the fine
control of live cell adhesion on the surface of natural or biomimetic
scaffolds. Therefore, the quantitative and precise measurement of the adhesion
strength of living cells is critical, not only in basic research but in modern
technologies, too. Several techniques have been developed or are under
development to quantify cell adhesion. All of them have their pros and cons,
which has to be carefully considered before the experiments and interpretation
of the recorded data. Current review provides a guide to choose the appropriate
technique to answer a specific biological question or to complete a biomedical
test by measuring cell adhesion
Classification of analytics, sensorics, and bioanalytics with polyelectrolyte multilayer capsules
Polyelectrolyte multilayer (PEM) capsules, constructed by LbL (layer-by-layer)-adsorbing polymers on sacrificial templates, have become important carriers due to multifunctionality of materials adsorbed on their surface or encapsulated into their interior. They have been also been used broadly used as analytical tools. Chronologically and traditionally, chemical analytics has been developed first, which has long been synonymous with all analytics. But it is not the only development. To the best of our knowledge, a summary of all advances including their classification is not available to date. Here, we classify analytics, sensorics, and biosensorics functionalities implemented with polyelectrolyte multilayer capsules and coated particles according to the respective stimuli and application areas. In this classification, three distinct categories are identified: (I) chemical analytics (pH; K+, Na+, and Pb2+ ion; oxygen; and hydrogen peroxide sensors and chemical sensing with surface-enhanced Raman scattering (SERS)); (II) physical sensorics (temperature, mechanical properties and forces, and osmotic pressure); and (III) biosensorics and bioanalytics (fluorescence, glucose, urea, and protease biosensing and theranostics). In addition to this classification, we discuss also principles of detection using the above-mentioned stimuli. These application areas are expected to grow further, but the classification provided here should help (a) to realize the wealth of already available analytical and bioanalytical tools developed with capsules using inputs of chemical, physical, and biological stimuli and (b) to position future developments in their respective fields according to employed stimuli and application areas
Polarized cortical tension drives zebrafish epiboly movements
The principles underlying the biomechanics of morphogenesis are
largely unknown. Epiboly is an essential embryonic event in which
three tissues coordinate to direct the expansion of the blastoderm.
How and where forces are generated during epiboly, and how
these are globally coupled remains elusive. Here we developed a
method, hydrodynamic regression (HR), to infer 3D pressure fields,
mechanical power, and cortical surface tension profiles. HR is
based on velocity measurements retrieved from 2D+T microscopy
and their hydrodynamic modeling. We applied HR to identify
biomechanically active structures and changes in cortex local
tension during epiboly in zebrafish. Based on our results, we
propose a novel physical description for epiboly, where tissue
movements are directed by a polarized gradient of cortical tension.
We found that this gradient relies on local contractile forces at the
cortex, differences in elastic properties between cortex components
and the passive transmission of forces within the yolk cell.
All in all, our work identifies a novel way to physically regulate
concerted cellular movements that might be instrumental for the
mechanical control of many morphogenetic processes.Peer ReviewedPostprint (author's final draft
Nano handling and measurement of biological cells in culture
A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of PhilosophyThis thesis systematically investigates the nano handling and measurement techniques for biological cells in culture and studies the techniques to realize innovative and multi-functional applications in biomedicine. Among them, the technique based on AFM is able to visualize and quantify the dynamics of organic cells in culture on the nano scale. Especially, the cellular shear adhesion force on the various locations of biological cells was firstly accurately measured in the research of the cell-substrate interaction in terms of biophysical perspective. The innovative findings are conductive to study the cell-cell adhesion, the cell-matrix adhesion which is related to the cell morphology structure, function, deformation ability and adhesion of cells and better understand the cellular dynamic behaviors. Herein, a new liquid-AFM probe unit and an increment PID control algorithm were implemented suitable for scanning the cell samples under the air conditions and the liquid environments. The influence between the surface of sample and
the probe, and the damage of probe during the sample scanning were reduced. The proposed system is useful for the nano handling and measurement of living cells.
Besides, Besides, to overcome the limitations of liquid-AFMs, the multiple optical tweezers were developed to integrate with the liquid-AFM. The technique based on laser interference is able to characterize the optical trap stiffness and the escape velocity, especially to realize the capture and sorting of multiple cells by a polarization-controlled periodic laser interference. It can trap and move hundreds of cells without physical contact, and has broad application prospects in cytology. Herein, a new experimental method integrated with the positioning analysis in the Z direction was used to improve the fluid force method for the calibration and characterize the mechanical forces exerted on optical traps and living cells. Moreover, a sensitive and highly efficient polarization-controlled three-beam interference set-up was developed for the capture and sorting of multiple cells. By controlling the polarization angles of the beams, various intensity distributions and different sizes of dots were obtained. Subsequently, we have experimentally observed multiple optical tweezers and the sorting of cells with different polarization angles, which are in accordance with the theoretical analysis
Measuring cell adhesion forces with the atomic force microscope at the molecular level
In the past 25 years many techniques have been developed to characterize cell adhesion and to quantify adhesion forces. Atomic force microscopy (AFM) has been used to measure forces in the pico-newton range, an experimental technique known as force spectroscopy. We modified such an AFM to measure adhesion forces between live cells or between cells and surfaces. This strategy required functionalizing the surface of the sensors for immobilizing the cell. We used Dictyostelium discoideum cells which respond to starvation by surface expression of the adhesion molecule csA and consequent aggregation to measure the adhesion force of a single csA-csA bond. Relevant experimental parameters include the duration of contact between the interacting surfaces, the force against which this contact is maintained, the number and specificity of interacting adhesion molecules and the constituents of the medium in which the interaction occurs. This technology also permits the measurement of the viscoelastic properties of single cells or cell layers. Copyright (C) 2002 S, Karger AG, Basel
Power laws in microrheology experiments on living cells: comparative analysis and modelling
We compare and synthesize the results of two microrheological experiments on
the cytoskeleton of single cells. In the first one, the creep function J(t) of
a cell stretched between two glass plates is measured after applying a constant
force step. In the second one, a micrometric bead specifically bound to
transmembrane receptors is driven by an oscillating optical trap, and the
viscoelastic coefficient is retrieved. Both and
exhibit power law behavior: and , with the same exponent
. This power law behavior is very robust ; is
distributed over a narrow range, and shows almost no dependance on the cell
type, on the nature of the protein complex which transmits the mechanical
stress, nor on the typical length scale of the experiment. On the contrary, the
prefactors and appear very sensitive to these parameters. Whereas
the exponents are normally distributed over the cell population, the
prefactors and follow a log-normal repartition. These results are
compared with other data published in the litterature. We propose a global
interpretation, based on a semi-phenomenological model, which involves a broad
distribution of relaxation times in the system. The model predicts the power
law behavior and the statistical repartition of the mechanical parameters, as
experimentally observed for the cells. Moreover, it leads to an estimate of the
largest response time in the cytoskeletal network: s.Comment: 47 pages, 14 figures // v2: PDF file is now Acrobat Reader 4 (and up)
compatible // v3: Minor typos corrected - The presentation of the model have
been substantially rewritten (p. 17-18), in order to give more details -
Enhanced description of protocols // v4: Minor corrections in the text : the
immersion angles are estimated and not measured // v5: Minor typos corrected.
Two references were clarifie
High performance, LED powered, waveguide based total internal reflection microscopy.
Total internal reflection fluorescence (TIRF) microscopy is a rapidly expanding optical technique with excellent surface sensitivity and limited background fluorescence. Commercially available TIRF systems are either objective based that employ expensive special high numerical aperture (NA) objectives or prism based that restrict integrating other modalities of investigation for structure-function analysis. Both techniques result in uneven illumination of the field of view and require training and experience in optics. Here we describe a novel, inexpensive, LED powered, waveguide based TIRF system that could be used as an add-on module to any standard fluorescence microscope even with low NA objectives. This system requires no alignment, illuminates the entire field evenly, and allows switching between epifluorescence/TIRF/bright field modes without adjustments or objective replacements. The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes
The Effect of Biomechanical and Biochemical Factors on Endothelial Cells: Relevance to Atherosclerosis
Microscale technologies create great opportunities for biologists to unveil cellular or molecular mechanisms of complex biological processes. Advanced measuring techniques, like atomic force microscope (AFM), allow detecting and controlling biological samples at high spatial and temporal resolution. Further integration with microsystems, such as microfluidic platforms, gives the ability to get detailed insight into basic biological phenomena. Highly integrated microdevices show great promise for biomedical research and potential clinical applications.
It is hypothesized that biomechanical factors play a significant role in the development of vascular diseases like atherosclerosis. To explore effects of biomechanical and biochemical stimuli on endothelial cells (ECs), AFM, which allows measurements of living cells, was utilized. Due to the heterogeneity of cells, standard characterization methods for mechanical properties of cells are still lacking. Therefore, a new quantitative method was developed for evaluation of cell elasticity correlating with cell morphology in this study. Moreover, cells are intrinsically viscoelastic materials revealed by stress relaxation measurements. A mechanically distinct bilayer model was proposed to discover the mechanical behaviour of cell components. Based on the elasticity characterization method and the stress relaxation model, the effect of cholesterol content on the mechanical response of ECs was examined, focusing on the behaviour of plasma membrane.
To mimic physiological conditions more closely for in vitro settings, a mask-free, highly integrated, low cost and time effective method was developed to rapidly fabricate a prototype of microfluidic cell culture system (MCCS). To better understand cell-cell interaction in circulatory systems like MCCS, a theoretical study of evaluating intercellular forces was also performed. Based on MCCS and microvalve technique, a novel bio-inspired and cell-based system was developed to simulate the formation of atherosclerosis plaque. Biomechanical properties of ECs, hemodynamic effects, cell rolling and adhesion events were investigated under this pathological model. The devices can be leveraged for potential applicability to biological research and clinical tests such as drug screening.
This research project has led to a better understanding of the underlying mechanisms of atherosclerosis and mechanical behaviours of ECs, as well as the development of AFM-based models that will be useful in determining cellular mechanical properties
Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS
Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease
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