Measuring molecular elasticity by atomic force microscope cantilever fluctuations

In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.     

Indentation with atomic force microscope,Saccharomyces cerevisiae cell gains elasticity under ethanol stress

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane is the first target to be attacked by the accumulated ethanol. In such a prominent position, S. cerevisiae cell membrane could reversely provide protection through changing fluidity or elasticity secondary to remodeled membrane components or structure during the fermentation process. However, there is yet to be a direct observation of the real effect of the membrane compositional change. In this study, atomic force microscope-based strategy was performed to determine Young's modulus of S. cerevisiae to directly clarify ethanol stress-associated changes and roles of S. cerevisiae cell membrane fluidity and elasticity. Cell survival rate decreased while the cell swelling rate and membrane permeability increased as ethanol concentration increased from 0% to 20% v/v. Young's modulus decreased continuously from 3.76 MPa to 1.53 MPa while ethanol stress increased from 0% to 20% v/v, indicating that ethanol stress induced the S. cerevisiae membrane fluidity and elasticity changes. Combined with the fact that membrane composition varies under ethanol stress, to some extent, this could be considered as a forced defensive act to the ethanol stress by S. cerevisiae cells. On the other hand, the ethanol stress induced loosening of cell membrane also caused S. cerevisiae cell to proactively remodel membrane to make cell membrane more agreeable to the increase of environmental threat. Increased ethanol stress made S. cerevisiae cell membrane more fluidized and elastic, and eventually further facilitated yeast cell’s survival.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Imaging viscoelasticity by force modulation with the atomic force microscope

Force modulation and phase sensitive detection was used to image soft surfaces with the atomic force microscope. This force modulation microscopy allows the simultaneous recording of images of the surface profile, the storage modulus, and the loss modulus of the sample. A theoretical treatment of the elastic tip-sample interaction is given. As examples, images of Langmuir-Blodgett films of a polymeric amphiphile and of a structured fatty acid are presented.     

Imaging cells with the atomic force microscope

Different types of cells have been imaged with the atomic force microscope. The morphology of the archaebacterium Halobacterium halobium in its dry state was revealed. On a leaf of the small Indian tree Lagerstroemia subcostata a stoma was imaged. The lower side of a water lily leaf was imaged in water showing features down to 12 nm. Finally, fixed red and white blood cells were imaged in buffer showing features down to 8 nm. The images demonstrate that atomic force microscopy can provide high-resolution images of cell surfaces under physiological conditions.     

Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation

The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.     

难溶性药用纳米微粒的原子力显微检测

建立用原子力显微镜(AFM)检测难溶性药用纳米微粒的方法。将纳米微粒配成一定浓度的悬浮液,以新鲜裂解的云母表面作为样品载体,在室温下用AFM的接触模式成像,用粒度分析软件进行粒度分析。以此方法对两种微粒(活性炭及氧化锌纳米微粒)进行了成像和测量,同时对两种样品进行透射电镜(TEM)观察。AFM检测结果显示,纳米活性炭微粒形态近似球形,表面较平滑,平均直径为(299±187)nm;纳米氧化锌微粒呈球形,平均直径(50±20)nm,与TEM检测结果具有较高一致性。该法简便可靠。     

Quantitative membrane electrostatics with the atomic force microscope

The atomic force microscope (AFM) is sensitive to electric double layer interactions in electrolyte solutions, but provides only a qualitative view of interfacial electrostatics. We have fully characterized silicon nitride probe tips and other experimental parameters to allow a quantitative electrostatic analysis by AFM, and we have tested the validity of a simple analytical force expression through numerical simulations. As a test sample, we have measured the effective surface charge density of supported zwitterionic dioleoylphosphatidylcholine membranes with a variable fraction of anionic dioleoylphosphatidylserine. The resulting surface charge density and surface potential values are in quantitative agreement with those predicted by the Gouy-Chapman-Stern model of membrane charge regulation, but only when the numerical analysis is employed. In addition, we demonstrate that the AFM can detect double layer forces at a separation of several screening lengths, and that the probe only perturbs the membrane surface potential by <2%. Finally, we demonstrate 50-nm resolution electrostatic mapping on heterogeneous model membranes with the AFM. This novel combination of capabilities demonstrates that the AFM is a unique and powerful probe of membrane electrostatics.     

应用原子力显微镜分析猪脂肪前体细胞的分化

脂肪形成过程中发生的异常变化与许多疾病的产生有着密切的关系。为深入了解脂肪形成的机制,利用原子力显微镜研究脂肪前体细胞向成熟脂肪细胞分化前后细胞形貌、超微结构和机械性能的变化。结果表明,脂肪前体细胞与成熟脂肪细胞在形貌上存在明显的差异。在超微结构的探测中成熟脂肪细胞表面粗糙度低于脂肪前体细胞。通过力曲线的分析得出,分化前后两种细胞的机械性质均发生改变。脂肪前体细胞在粘弹力、硬度和杨氏模量的研究中比成熟脂肪细胞都高出约20％。原子力显微镜在纳米尺度上分析脂肪前体细胞向成熟脂肪细胞分化过程中细胞膜的改变,其研究结果为进一步探讨脂肪形成机制提供可视化定量数据。     

原子力显微镜研究小鼠免疫球蛋白G自组装

应用原子力显微镜观察小鼠免疫球蛋白G样品,结果不管是IgG1还是IgG1与IgG2的混合样品,在制样2-3d后,均发现有圆环状聚集区,也有部分圆面状聚集区域,一般环中心有核,核为IgG分子多聚体的再聚集,多聚体中亚基（单体）数目因环而异,在圆环和贺湎状区域外,也有零星分散的聚集体,不参与成环,另外,IgG1和IgG1 IgG2聚集情况有所不同,后者更倾向于形成圆面状聚集,另外,在聚集发生前,亚基已自组装形成各种形状的多聚体,然后多聚体进一步聚集形成各种圆环或圆面状,对免疫球蛋白G先自组装而后聚集的过程,从生物学,物理学角度作了初步解释。     

Biomolecular force measurements and the atomic force microscope

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.     

Living under an atomic force microscope

An approach for long‐term in vivo investigations on cyanobacterial cell surface changes at high spatial resolution by Atomic Force Microscopy (AFM) was developed in this study. Until recently, changes of bacterial cell surfaces due to changes of the chemical environment could neither be investigated in situ nor in vivo. However, in vivo investigations give insights into kinetics of cell response to environmental changes and mineral nucleation at the cell's surface. Continuously cultured cyanobacteria of the representative freshwater strain Synechococcus leopoliensis (PCC 7942) were washed and artificially immobilized on poly‐l‐lysine‐coated glass slides. Both immobilization and environmental conditions were optimized in order to facilitate long‐term experiments (> 100 h) with living cells. AFM samples were investigated in situ in two different solutions: Culture medium was used for cultivation experiments and nutrient‐free NaHCO₃/CaCl₂ solutions (supersaturated with respect to calcite) for long‐term characterizations of the changes in cell surface topography. Cell viability under these conditions was investigated by AFM, TEM and epifluorescence microscopy, independently. No indications for extended starvation were found within the relevant timescales. Analysing the influence of Ca²⁺ on the surface of S. leopoliensis, we found significant changes compared to a Ca‐free solution. Few hours after CaCl₂ was added to the circumfluent solution, small protuberances were observed on the cell surface. These are promising results to environmental scientists for a wide range of applications, as cell response to environmental changes can now be monitored online and in vivo at timescales, which are relevant for natural processes. Most especially studies of biomineralization and mineral nucleation on bacterial cell surfaces will profit from this new approach.     

Amyloid under the atomic force microscope

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.     

Mapping interaction forces with the atomic force microscope.

Force curves were recorded as the sample was raster-scanned under the tip. This opens new opportunities for imaging with the atomic force microscope: several characteristics of the samples can be measured simultaneously, for example, topography, adhesion forces, elasticity, van der Waals, and electrostatic interactions. The new opportunities are illustrated by images of several characteristics of thin metal films, aggregates of lysozyme, and single molecules of DNA.     

Sensing specific molecular interactions with the atomic force microscope

One of the unique features of the atomic force microscope (AFM) is its capacity to measure interactions between tip and sample with high sensitivity and unparalleled spatial resolution. Since the development of methods for the functionalization of the tips, the versatility of the AFM has been expanded to experiments where specific molecular interactions are measured. For illustration, we present measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. Here, we compare different techniques that allow for the in situ measurement of the absolute value of the spring constant of cantilevers.     

Visualization of hemiknot DNA structure with an atomic force microscope

The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)_n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane–mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.     

Erythrocyte stiffness probed using atomic force microscope

The stiffness of erythrocytes in patients (N=45) suffering from certain disorders, such as coronary disease, hypertension, and diabetes mellitus has been assessed using the Atomic Force Microscopy (AFM) and compared with that in a group of healthy individuals (N=13). For each blood sample, around 20 erythrocytes were selected at random and the stiffness of each one was probed in 20-30 arbitrarily chosen points. From these results, distributions of the cell Young's modulus (YM) were determined. Average values and widths of YM distributions significantly increased in samples taken from diabetes mellitus patients and cigarette smokers, as compared to those taken from healthy donors. At the same time, the average values of YM were found to increase as a function of the patient's age. We demonstrated that the atomic force microscope is a very sensitive tool for determination of cell stiffness with every prospect of a routine application as a diagnostic tool in quantitative analysis of the physiological and pathological states of red blood cells.     

Imaging biological structures with the cryo atomic force microscope.

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.     

Observing single biomolecules at work with the atomic force microscope

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.