Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

Nanomechanical insights: Amyloid beta oligomer-induced senescent brain endothelial cells

Senescent cells accumulate in various peripheral tissues during aging and have been shown to exacerbate age-related inflammatory responses. We recently showed that exposure to neurotoxic amyloid β (Aβ1–42) oligomers can readily induce a senescence phenotype in human brain microvascular endothelial cells (HBMECs). In the present work, we used atomic force microscopy (AFM) to further characterize the morphological properties such as cell membrane roughness and cell height and nanomechanical properties such as Young's modulus of the membrane (membrane stiffness) and adhesion resulting from the interaction between AFM tip and cell membrane in Aβ1–42 oligomer-induced senescent human brain microvascular endothelial cells. Morphological imaging studies showed a flatter and spread-out nucleus in the senescent HBMECs, both characteristic features of a senescent phenotype. Furthermore, the mean cell body roughness and mean cell height were lower in senescent HBMECs compared to untreated normal HBMECs. We also observed increased stiffness and alterations in the adhesion properties in Aβ1–42 oligomer-induced senescent endothelial cells compared to the untreated normal HBMECs suggesting dynamic reorganization of cell membrane. We then show that vascular endothelial growth factor receptor 1 (VEGFR-1) knockdown or overexpression of Rho GTPase Rac 1 in the endothelial cells inhibited senescence and reversed these nanomechanical alterations, confirming a direct role of these pathways in the senescent brain endothelial cells. These results illustrate that nanoindentation and topographic analysis of live senescent brain endothelial cells can provide insights into cerebrovascular dysfunction in neurodegenerative diseases such as Alzheimer's disease.     

Erythrocyte death in vitro induced by starvation in the absence of Ca

Human erythrocytes (RBCs), stored at 4 °C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R_rms) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4 days of storage at 4 °C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5 days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R_rms - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (≥ 90%) depleted in an irreversible way of the energetic stores.     

Oxygenation-deoxygenation cycle of erythrocytes modulates submicron cell membrane fluctuations.

Low frequency submicron fluctuations of the cell membrane were recently shown to be characteristic for different cell types, nevertheless their physiological role is yet unknown. Point dark-field microscopy based recordings of these local displacements of cell membrane in human erythrocytes, subjected to cyclic oxygenation and deoxygenation, reveals a reversible decrease of displacement amplitudes from 290 +/- 49 to 160 +/- 32 nm, respectively. A higher rate of RBC adhesion to a glass substratum is observed upon deoxygenation, probably due to a low level of fluctuation amplitudes. The variation in the amplitude of these displacements were reconstituted in open RBC ghosts by perfusing them with composite solutions of 2,3 diphosphoglycerate, Mg+2, and MgATP, which mimic the intracellular metabolite concentrations in oxygenated and deoxygenated erythrocytes. The mere change in intracellular Mg+2 during oxygenation-deoxygenation cycle is sufficient to explain these findings. The results imply that the magnitude of fluctuations amplitude is directly connected with cell deformability. This study suggests that the physiological cycle of oxygenation-deoxygenation provides a dynamic control of the bending deformability and adhesiveness characteristics of the RBC via a Mg+2-dependent reversible assembly of membrane-skeleton proteins. The existing coupling between oxygenation-deoxygenation of the RBC and its mechanical properties is expected to play a key role in blood microcirculation and may constitute an example of a general situation for other circulating blood cells, where the metabolic control of cytoskeleton dynamics may modulate their dynamic mechanical properties.     

Investigation of red blood cell mechanical properties using AFM indentation and coarse-grained particle method

Background

Red blood cells (RBCs) deform significantly and repeatedly when passing through narrow capillaries and delivering dioxygen throughout the body. Deformability of RBCs is a key characteristic, largely governed by the mechanical properties of the cell membrane. This study investigated RBC mechanical properties using atomic force microscopy (AFM) with the aim to develop a coarse-grained particle method model to study for the first time RBC indentation in both 2D and 3D. This new model has the potential to be applied to further investigate the local deformability of RBCs, with accurate control over adhesion, probe geometry and position of applied force.

Results

The model considers the linear stretch capacity of the cytoskeleton, bending resistance and areal incompressibility of the bilayer, and volumetric incompressibility of the internal fluid. The model’s performance was validated against force–deformation experiments performed on RBCs under spherical AFM indentation. The model was then used to investigate the mechanisms which absorbed energy through the indentation stroke, and the impact of varying stiffness coefficients on the measured deformability. This study found the membrane’s bending stiffness was most influential in controlling RBC physical behaviour for indentations of up to 200 nm.

Conclusions

As the bilayer provides bending resistance, this infers that structural changes within the bilayer are responsible for the deformability changes experienced by deteriorating RBCs. The numerical model presented here established a foundation for future investigations into changes within the membrane that cause differences in stiffness between healthy and deteriorating RBCs, which have already been measured experimentally with AFM.

     

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.     

Atomic force pulling: probing the local elasticity of the cell membrane

We present a novel approach, based on atomic force microscopy, for exploring the local elastic properties of the membrane-skeleton complex in living cells. Three major elements constitute the basis for the proposed method: (1) pulling the cell membrane by increasing the adhesion of the tip to the cell surface provided via appropriate tip modification; (2) measuring force-distance curves with emphasis on selecting the appropriate withdrawal regions for analysis; (3) fitting of the theoretical model for axisymmetric bending of an annular thick plate to the experimental curve in the withdrawal region, prior to the detachment point of the tip from the cell membrane. This approach, applied to human erythrocytes, suggests a complimentary technique to the commonly used methods. The local use of this methodology for determining the bending modulus of the cell membrane of the human erythrocyte yields a value of (2.07+/-0.32) x 10(-19) J.     

Mechanical compression insults induce nanoscale changes of membrane-skeleton arrangement which could cause apoptosis and necrosis in dorsal root ganglion neurons

In a primary spinal cord injury, the amount of mechanical compression insult that the neurons experience is one of the most critical factors in determining the extent of the injury. The ultrastructural changes that neurons undergo when subjected to mechanical compression are largely unknown. In the present study, using a compression-driven instrument that can simulate mechanical compression insult, we applied mechanical compression stimulation at 0.3, 0.5, and 0.7?MPa to dorsal root ganglion (DRG) neurons for 10?min. Combined with atomic force microscopy, we investigated nanoscale changes in the membrane-skeleton, cytoskeleton alterations, and apoptosis induced by mechanical compression injury. The results indicated that mechanical compression injury leads to rearrangement of the membrane-skeleton compared with the control group. In addition, mechanical compression stimulation induced apoptosis and necrosis and also changed the distribution of the cytoskeleton in DRG neurons. Thus, the membrane-skeleton may play an important role in the response to mechanical insults in DRG neurons. Moreover, sudden insults caused by high mechanical compression, which is most likely conducted by the membrane-skeleton, may induce necrosis, apoptosis, and cytoskeletal alterations.     

Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton

We investigate the mechanical strength of adhesion and the dynamics of detachment of the membrane from the cytoskeleton of red blood cells (RBCs). Using hydrodynamical flows, we extract membrane tethers from RBCs locally attached to the tip of a microneedle. We monitor their extrusion and retraction dynamics versus flow velocity (i.e., extrusion force) over successive extrusion-retraction cycles. Membrane tether extrusion is carried out on healthy RBCs and ATP-depleted or -inhibited RBCs. For healthy RBCs, extrusion is slow, constant in velocity, and reproducible through several extrusion-retraction cycles. For ATP-depleted or -inhibited cells, extrusion dynamics exhibit an aging phenomenon through extrusion-retraction cycles: because the extruded membrane is not able to retract properly onto the cell body, each subsequent extrusion exhibits a loss of resistance to tether growth over the tether length extruded at the previous cycle. In contrast, the additionally extruded tether length follows healthy dynamics. The extrusion velocity L depends on the extrusion force f according to a nonlinear fashion. We interpret this result with a model that includes the dynamical feature of membrane-cytoskeleton association. Tether extrusion leads to a radial membrane flow from the cell body toward the tether. In a distal permeation regime, the flow passes through the integral proteins bound to the cytoskeleton without affecting their binding dynamics. In a proximal sliding regime, where membrane radial velocity is higher, integral proteins can be torn out, leading to the sliding of the membrane over the cytoskeleton. Extrusion dynamics are governed by the more dissipative permeation regime: this leads to an increase of the membrane tension and a narrowing of the tether, which explains the power law behavior of L(f). Our main result is that ATP is necessary for the extruded membrane to retract onto the cell body. Under ATP depletion or inhibition conditions, the aging of the RBC after extrusion is interpreted as a perturbation of membrane-cytoskeleton linkage dynamics.     

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: a quantitative atomic force microscopy investigation

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.     

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt??s lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young??s modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young??s modulus than that of indolent cancer cells, which may improve our understanding of metastasis.     

Morphological and Physical Analysis of Natural Phospholipids-Based Biomembranes

Background

Liposomes are currently an important part of biological, pharmaceutical, medical and nutritional research, as they are considered to be among the most effective carriers for the introduction of various types of bioactive agents into target cells.

Scope of Review

In this work, we study the lipid organization and mechanical properties of biomembranes made of marine and plant phospholipids. Membranes based on phospholipids extracted from rapeseed and salmon are studied in the form of liposome and as supported lipid bilayer. Dioleylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) are used as references to determine the lipid organization of marine and plant phospholipid based membranes. Atomic force microscopy (AFM) imaging and force spectroscopy measurements are performed to investigate the membranes'' topography at the micrometer scale and to determine their mechanical properties.

Major Conclusions

The mechanical properties of the membranes are correlated to the fatty acid composition, the morphology, the electrophoretic mobility and the membrane fluidity. Thus, soft and homogeneous mechanical properties are evidenced for salmon phospholipids membrane containing various polyunsaturated fatty acids. Besides, phase segregation in rapeseed membrane and more important mechanical properties were emphasized for this type of membranes by contrast to the marine phospholipids based membranes.

General Significance

This paper provides new information on the nanomechanical and morphological properties of membrane in form of liposome by AFM. The originality of this work is to characterize the physico-chemical properties of the nanoliposome from the natural sources containing various fatty acids and polar head.     

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: A quantitative atomic force microscopy investigation

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.     

Nanoscale strain-hardening of keratin fibres

Mammalian appendages such as hair, quill and wool have a unique structure composed of a cuticle, a cortex and a medulla. The cortex, responsible for the mechanical properties of the fibers, is an assemblage of spindle-shaped keratinized cells bound together by a lipid/protein sandwich called the cell membrane complex. Each cell is itself an assembly of macrofibrils around 300 nm in diameter that are paracrystalline arrays of keratin intermediate filaments embedded in a sulfur-rich protein matrix. Each macrofibril is also attached to its neighbors by a cell membrane complex. In this study, we combined atomic force microscopy based nano-indentation with peak-force imaging to study the nanomechanical properties of macrofibrils perpendicular to their axis. For indentation depths in the 200 to 500 nm range we observed a decrease of the dynamic elastic modulus at 1 Hz with increasing depth. This yielded an estimate of 1.6GPa for the lateral modulus at 1 Hz of porcupine quill's macrofibrils. Using the same data we also estimated the dynamic elastic modulus at 1 Hz of the cell membrane complex surrounding each macrofibril, i.e., 13GPa. A similar estimate was obtained independently through elastic maps of the macrofibrils surface obtained in peak-force mode at 1 kHz. Furthermore, the macrofibrillar texture of the cortical cells was clearly identified on the elasticity maps, with the boundaries between macrofibrils being 40-50% stiffer than the macrofibrils themselves. Elasticity maps after indentation also revealed a local increase in dynamic elastic modulus over time indicative of a relaxation induced strain hardening that could be explained in term of a α-helix to β-sheet transition within the macrofibrils.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

原子力显微镜研究不同刺激条件下T细胞的形态和生物力学特性

T细胞的抗原识别和活化可以直接影响整个免疫应答的性质、效能和结果,在人体免疫反应中具有核心作用.细胞的形态结构和力学特性决定着细胞的功能的发挥.利用原子力显微镜（AFM）从纳米水平和皮牛顿量级探测分析静息的T细胞和不同刺激剂（超抗原SEA和植物凝集素PHA）活化的T细胞的形态结构和生物力学特性.研究发现静息的T细胞呈较为规则的圆形,细胞表面相对光滑均一,活化后细胞高度和体积明显增大,体积增大为静息T细胞的2~3倍,高度增加了约50%,这是T细胞经过刺激剂活化后增殖、分化而增大的表现.同时发现活化后的T细胞表面粗糙度增大,细胞表面形成100 nm~1 μm颗粒状团簇结构.这种微纳结构域的形成与T细胞经过活化后细胞表面分子表达和细胞因子的分泌有关,并且与免疫突触的形成和功能发挥密切关联.经过PHA和SEA活化后的T细胞表面粘附力增大,是静息的T细胞的3-6倍,而细胞硬度明显减小,这种力学特性的变化有利于T细胞与病原体的相关作用从而清除病原体.通过AFM的研究,可以进一步的了解T淋巴细胞形态变化与细胞行为之间的关系,为更好地理解T细胞的结构与功能提供了更多可视化的依据.     

Disulfide bonds in the outer layer of keratin fibers confer higher mechanical rigidity: correlative nano-indentation and elasticity measurement with an AFM.

Nanomechanical properties of biological fibers are governed by the morphological features and chemically heterogeneous constituent subunits. However, very little experimental data exist for nanoscale correlation between heterogeneous subunits and their mechanical properties. We have used keratin-rich wool fibers as a model of composite biological fibers; a wool fiber is a simple two component cylindrical system consisting of a core cellular component surrounded by an outer cell layer and their ultrastructure and chemical composition are well-characterized. The core is 16-40 micrometer in diameter and rich in axially aligned keratin microfibrils. Outer cells have multiple laminar layers, 60-600 nm thick and distinctly rich in disulfide bonds. We used an atomic force microscope (AFM) to examine the nanomechanical properties of various structural components using complementary techniques of force-volume imaging and nano-indentation. AFM images of transverse sections of fibers were obtained in ambient environment, and the mechanical properties of several identified regions were examined. The outer cell layer showed a significantly higher mechanical stiffness than the internal cellular core region. Chemical reduction of disulfide bonds eliminated such dichotomy of mechanical strengths, indicating that the higher rigidity of the outer layer is attributed primarily to the presence of extensive disulfide bonding in the exo-cuticle. This is the first detailed correlative study of nano-indentation and regional elasticity measurements in composite biological systems, including mammalian biological fibers.