Detection of human erythrocytes influenced by iron deficiency anemia and thalassemia using atomic force microscopy

PurposeIron deficiency anemia (IDA) and thalassemia (THAL) are two types of microcytic anemia, and both of these conditions disturb the morphology and function of erythrocytes at the molecular level. The ability to distinguish between thalassemic and iron deficiency anemia microcytosis has important clinical implications. The purpose of this study was to show that pathophysiological changes to erythrocytes could be detected at the nanometer scale, which is important for the early diagnosis of anemia and for distinguishing between IDA and THAL.MethodsBlood samples were taken from six healthy volunteers and six patients with either iron deficiency anemia or thalassemia. Changes in the morphological of erythrocytes were studied at the nanometer level using atomic force microscopy (AFM).ResultsThere were dramatic overall shape and surface membrane deformations of the erythrocytes associated with iron deficiency anemia and thalassemia compared to healthy erythrocytes. Healthy and pathological erythrocytes of iron deficiency anemia and thalassemia could be distinguished by the morphologic parameters of width, length, the ratio of length to width, valley, peak, valley-to-peak, standard deviations, and surface fluctuation. These AFM parameters of erythrocyte morphology differed greatly between iron deficiency anemia and thalassemia.ConclusionAFM was found to be an extremely useful tool for detecting and distinguishing between iron deficiency anemia and thalassemia. Erythrocyte morphology is an important determinant for diagnosing and distinguishing IDA and THAL.     

Investigation of nano-mechanical properties of annulus fibrosus using atomic force microscopy

We describe the use of atomic force microscopy (AFM) to investigate the nanomechanical properties of annulus fibrosus (AF)-the outer fibrous layer of an intervertebral disc (IVD) encapsulating the inner jelly-like mass known as the nucleus pulposus (NP). Disk disease, degenerated discs, slipped discs, and herniated discs are common terms often linked to back pain and are caused due to degeneration of IVD. Due to the variations in the structure and biochemical composition of the IVD, studies of macromechanical properties in the motion segment or AF may lack all significant nanomechanical responses or behaviors. Existing studies do not report the micro or nano level of mechanics of IVD components and whether the nanomechanics of this tissue mimic its macromechanical behavior is not known. Our studies used AFM to investigate the regional micromechanical properties of the AF that have been otherwise difficult due to small sample size of the tissue. Five different zones including peripheral and central were tested mechanically as well as biochemically. Qualitative biochemical staining and quantitative values of nanomechanical properties of different zones are compared and discussed in detail. The results of nanomechanical investigations described in this study not only reveal its mimic at macroscopic level, they represent an important step towards establishing a framework for testing and comparing tissue engineered IVD replacements with native tissues.     

Surface investigation of adhesive formulation consisting of UV sensitive triblock poly(styrene–b-butadiene–b-styrene) copolymer

Atomic force microscopy (AFM) analysis in conjunction with macroscopic studies such as peel testing and contact angle measurement have been undertaken to explain the nanomechanical properties of adhesive formulation consisting of triblock poly(styrene–b-butadiene–b-styrene) (SBS) copolymers. The cross-linking of this photosensitive copolymer was investigated by analyzing the mechanical and morphological changes of each phase induced by the UV exposure. Main result is that the adhesive properties are strongly influenced by the cross-linking of the polybutadiene (PB) phase leading to an increase in the surface stiffness without affecting the surface energy. AFM analysis shows that the adhesion force is mostly governed by the contact area between the adhesive and the probe. The surface mobility may explain the increase in adhesion for this pressure sensitive copolymer.     

Atomic force microscopy of swelling and hardening of intact erythrocytes fixed on substrate

Peak force measurements with the aid of atomic force microscopy are used to quantitatively map nanomechanical properties of intact erythrocytes of rats under conditions that are close to physiological conditions. Erythrocytes that are immobilized on the substrate preliminary processed using poly-L-lysine predominantly exhibit plane shape. However, cells may also exhibit stepwise transformation to semispherical objects with an increase in volume and hardening. Possible reasons for such transformations are discussed.     

Atomic force microscopy probing of cell elasticity

Atomic force microscopy (AFM) has recently provided the great progress in the study of micro- and nanostructures including living cells and cell organelles. Modern AFM techniques allow solving a number of problems of cell biomechanics due to simultaneous evaluation of the local mechanical properties and the topography of the living cells at a high spatial resolution and force sensitivity. Particularly, force spectroscopy is used for mapping mechanical properties of a single cell that provides information on cellular structures including cytoskeleton structure.

This entry is aimed to review the recent AFM applications for the study of dynamics and mechanical properties of intact cells associated with different cell events such as locomotion, differentiation and aging, physiological activation and electromotility, as well as cell pathology. Local mechanical characteristics of different cell types including muscle cells, endothelial and epithelial cells, neurons and glial cells, fibroblasts and osteoblasts, blood cells and sensory cells are analyzed in this paper.     

Cortical rigidity of round cells in mitotic phase and suspended state

This paper describes the results of the analysis of cortical rigidity in two round cell states: mitotic round cells and detached round cells after trypsinization using atomic force microscopy (AFM). These two states are primary cell events with dynamic morphological alterations in vitro. The trypsinized detached cells were fixed on the substrate of membrane anchoring oleyl surface. Fluorescent images taken by confocal laser scanning microscopy revealed diverse cell surface protrusions and cortical actin development in the round cells under different conditions. Although the cortical actin of these cells seemed to develop similarly, cortical rigidity of the trypsinized round cells showed greater stiffness than that of mitotic round cells. The elasticity measurements by AFM may detect invisible information about the maturation or strength of F-actin structures and such measurements may indicate that the strength of the actomyosin cortex would be higher in trypsinized round cells compared to mitotic cells. The mechanical properties can help provide better insights into the characteristics of the actin cytoskeleton network in vicinity of cell surface during dynamic morphological alterations.     

Membrane deformation of unfixed erythrocytes in air with time lapse investigated by tapping mode atomic force microscopy

Estimation of the time of death is one of the most important problems for forensic medicine and law. Physical and chemical postmortem changes are evaluated together while estimating the time of death. The pattern analysis of antemortem and postmortem bloodstains is one of the important parameters for forensic science, and cellular changes of blood cells can be useful for the quantitative assessment of the time of death. In this study, by successively investigating erythrocytes exposed in air on mica for 5 days using tapping mode atomic force microscopy (TM-AFM), we observed deformation of whole cell and membrane surface of unfixed erythrocytes with time lapse. We found that the time-dependent cellular changes occurred after exposure of erythrocytes in air for several days. At 0.5 days of exposure, fissures and cell shrinkage were observed. At 2.5 days of exposure, the emergence of nanometer-scale protuberances were observed and these protuberances increased in number with increasing time. The changes of cell shape and cell membrane surface ultrastructure can be used to estimate the time of death. Futhermore, smear-induced abnormal erythrocytes and immunostained erythrocytes were observed here. The need for more precise research is indicated, such as the correlation of membrane changes to intervals of less than 0.5 day of air exposure, and use of various substrates in addition to mica, including glass, metals, fabrics, among others, on which the bloodstains appear in crime scenes. The results of this research demonstrate the efficacy of AFM as a potentially powerful analytical tool in forensic science.     

Elasticity and nanomechanical response of Aspergillus niger spores using atomic force microscopy

The elasticity and nanomechanical response of Aspergillus niger spores determined using atomic force microscopy (AFM) and nanoindentation are discussed. The force-displacement curve of the spore surfaces shows that the average surface roughness of spores was approximately 33 nm and that the adhesion force ranged from 9 to 28 nN. The Young's modulus of the A. niger spores ranged from 0.1 to 21.4 GPa and the hardness ranged from 0.01 to 0.17 GPa. The critical buckling load of the spore membrane is 290 μN.     

Nanomechanical characteristics of annealed Si/SiGe superlattices

In this study, the nanomechanical damage was investigated on the annealed Si/SiGe strained-layer superlattices (SLSs) deposited using an ultrahigh-vacuum chemical vapor deposition (UHVCVD). Nanoscratch, nanoindenter, atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques were used to determine the nanomechanical behavior of the SiGe films. With a constant force applied, greater hardness number and larger coefficients of friction (μ) were observed on the samples that had been annealed at 600 °C, suggesting that annealing of the Si/SiGe SLSs can induce greater shear resistance. AFM morphological studies of the Si/SiGe SLSs revealed that pile-up phenomena occurred on both sides of each scratch, with the formation of some pellets and microparticles. The Si/SiGe SLSs that had been subjected to annealing under various conditions exhibited significantly different features in their indentation results. Indeed, the TEM images reveal slight dislocation propagation in the microstructures. Thus, the hardness and elastic modulus can be increased slightly after annealing treatment because the existence of comparatively unstable microstructures. It is suggested that cracking phenomena dominate the damage cause of Si/SiGe SLSs.     

Improved aging performance of vapor phase deposited hydrophobic self-assembled monolayers

A hydrophobic self-assembled monolayer (SAM) of fluoro-octyl-trichloro-silane (FOTS) was deposited on silicon using a vapor phase technique. The aging of the hydrophobic layer was examined using water contact angle measurements. It has been found that while such monolayer films suffer from a loss of hydrophobicity with time, pre-immersion nitrogen annealing can significantly improve the aging characteristics of these monolayers. The effect of nitrogen annealing on the improved aging properties of SAM coatings has been investigated by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The hydrolytic stability and the effect of nitrogen annealing were studied by morphological evolution during immersion. A spontaneous formation of silane mounds on the surface of the monolayers was found by AFM. These mounds have been irreversibly transformed from initially uniform hydrophobic surface layers. It is highly probable that the compliance of these mounds can reasonably allow hydrophilic sites to be located around the mounds. Interestingly, the density of these mounds formation is very less on the annealed samples. XPS reveals a higher level of coverage by the N₂-annealed film due to agglomeration. A relative abundance of CF₃ and CF₂ moieties in the annealed film may explain the enhancement of the hydrophobicity as revealed by higher level of water contact angle. This hydrophobicity was found to be significantly stable in water. This novel finding explains the improved hydrophobic stability of FOTS monolayers as primarily a morpho-chemical effect that originates from the densification of the monolayers upon annealing.     

Quantification issues in the identification of nanoscale regions of homopolymers using modulus measurement via AFM nanoindentation

Since 1989, AFMs have been used to map the nanomechanical properties of surfaces using measurements such as force-distance curves. Quantification of the force and elastic parameters are critical to the nanomechanical analysis and positive identification of materials at the nanoscale, as well as for assessing behaviour at surfaces. In recent years, there have been AFM papers publishing “quantitative” values for the indentation modulus, however, many involved large uncertainties arising from the lack of calibration of key components, the use of manufacturers’ nominal values for these components or the use of incorrect models. This paper addresses the quantification issues in modulus measurement at surfaces for homogeneous materials using force-distance curves and how to do this with sufficient accuracy to identify materials at the nanoscale. We review the available theory and describe two routes to quantitative modulus measurement using both the AFM on its own and the AFM combined with a nanoindenter. The first involves the direct measurement of modulus using a fully calibrated instrument and allows depth analysis. The second uses indirect measurement through calibration by reference materials of known reduced modulus. For depth analysis by this second route, these reference moduli need to be known as a function of depth. We show that, using the second route, an unknown polymer may be analysed using the nanoindenter, its modulus determined and, providing the moduli of the polymers to be identified or distinguished differ by more than 20%, identified with 95% confidence. We recommend that users evaluate a set of reference samples using a traceable nanoindenter via the first route, and then use these to calibrate the AFM by the second route for identification of nano-regions using the AFM.     

品质因子可调的石墨烯纳米机械振子

纳米机械振子尺寸小,质量轻,可以用来制作探测力、质量等微小物理量的超灵敏探测器.石墨烯拥有质量轻、密度低和杨氏模量高等特性,被认为是制作纳米机械振子的理想材料.石墨烯纳米机械振子因其具有的谐振频率高、品质因子高和谐振频率可调性高等优势,近年来得到了人们的广泛关注.作为表征纳米机械振子性能的一个重要指标,品质因子越高,意味着纳米机械振子耗散越低,纳米机械振子的灵敏度越高.本文通过微纳加工的工艺制备出一种谐振频率随栅压可调(调节的范围为73MHz~90MHz)的石墨烯纳米机械振子样品,研究其在极低温高真空环境下的品质因子与栅极电压之间的关系.实验表明通过栅压调节振子的内部应力,能够使石墨烯纳米机械振子品质因子从220提高到1000.我们的结果为二维材料纳米机械振子的耗散研究提供了一种新的研究思路.     

Morphological phase diagram for lipid membrane domains with entropic tension

Circular domains in phase-separated lipid vesicles with symmetric leaflet composition commonly exhibit three stable morphologies: flat, dimpled, and budded. However, stable dimples (i.e., partially budded domains) present a puzzle since simple elastic theories of domain shape predict that only flat and spherical budded domains are mechanically stable in the absence of spontaneous curvature. We argue that this inconsistency arises from the failure of the constant surface tension ensemble to properly account for the effect of entropic bending fluctuations. Formulating membrane elasticity within an entropic tension ensemble, wherein tension represents the free energy cost of extracting membrane area from thermal bending of the membrane, we calculate a morphological phase diagram that contains regions of mechanical stability for each of the flat, dimpled, and budded domain morphologies.     

Structure and morphology of Cu/Ni film grown by electrodeposition method: A study of neutron reflectivity and AFM

We present detailed study of structure and interface morphology of an electrodeposited Cu/Ni film using X-ray diffraction, X-ray reflectivity, neutron reflectivity and atomic force microscopy (AFM) techniques. The crystalline structure of the film has been determined by X-ray diffraction, which suggest polycrystalline growth of the film. The depth profile of density in the sample has been obtained from specular X-ray and neutron reflectivity measurements. AFM image of the air-film interface shows that the surface is covered by globular islands of different sizes. The AFM height distribution of the surface clearly shows two peaks and the relief structure (islands) on the surface in the film, which can be treated as a quasi-two-level random rough surface structure. We have demonstrated that the detailed morphology of air-film interfaces, the quasi-two-level surface structure as well as morphology of the buried interfaces can be obtained from off-specular neutron reflectivity data. AFM and off-specular neutron reflectivity measurements also show that the morphologies of electrodeposited surface is distinctively different as compared to that of sputter-deposited surfaces in the sample.     

Influence of surface energy and relative humidity on AFM nanomechanical contact stiffness

The effects of surface functionality and relative humidity (RH) on nanomechanical contact stiffness were investigated using atomic force acoustic microscopy (AFAM), a contact scanned-probe microscopy (SPM) technique. Self-assembled monolayers (SAMs) with controlled surface energy were studied systematically in a controlled-humidity chamber. AFAM amplitude images of a micropatterned, graded-surface-energy SAM sample revealed that image contrast depended on both ambient humidity and surface energy. Quantitative AFAM point measurements indicated that the contact stiffness remained roughly constant for the hydrophobic SAM but increased monotonically for the hydrophilic SAM. To correct for this unphysical behavior, a viscoelastic damping term representing capillary forces between the tip and the SAM was added to the data analysis model. The contact stiffness calculated with this revised model remained constant with RH, while the damping term increased strongly with RH for the hydrophilic SAM. The observed behavior is consistent with previous studies of surface energy and RH behavior using AFM pull-off forces. Our results show that surface and environmental conditions can influence accurate measurements of nanomechanical properties with SPM methods such as AFAM.     

Atomic force microscopy for the investigation of molecular and cellular behavior

The present review details the methods used for the measurement of cells and their exudates using atomic force microscopy (AFM) and outlines the general conclusions drawn by the mechanical characterization of biological materials through this method. AFM is a material characterization technique that can be operated in liquid conditions, allowing its use for the investigation of the mechanical properties of biological materials in their native environments. AFM has been used for the mechanical investigation of proteins, nucleic acids, biofilms, secretions, membrane bilayers, tissues and bacterial or eukaryotic cells; however, comparison between studies is difficult due to variances between tip sizes and morphologies, sample fixation and immobilization strategies, conditions of measurement and the mechanical parameters used for the quantification of biomaterial response. Although standard protocols for the AFM investigation of biological materials are limited and minor differences in measurement conditions may create large discrepancies, the method is nonetheless highly effective for comparatively evaluating the mechanical integrity of biomaterials and can be used for the real-time acquisition of elasticity data following the introduction of a chemical or mechanical stimulus. While it is currently of limited diagnostic value, the technique is also useful for basic research in cancer biology and the characterization of disease progression and wound healing processes.     

Chiral surfaces and metal/ceramic heteroepitaxy in the Pt/SrTiO3(621) system

Low-energy electron diffraction (LEED), atomic force microscopy (AFM), and X-ray diffraction (XRD) have been used to investigate the structural and morphological character of a naturally chiral ceramic SrTiO₃(621) substrate and of Pt and Cu thin films deposited on its surface. AFM experiments showed that as-received chirally-oriented SrTiO₃(621) substrates display atomically smooth surface morphologies, while LEED patterns revealed that the surface structure has a net chirality. Pt(621) and Cu(621) thin films were grown heteroepitaxially on SrTiO₃(621) substrates, as confirmed by XRD. AFM showed that the film surfaces were atomically smooth and LEED illustrated that the Pt films exhibit surface chirality, and by implication that the atomically-flat chirally-oriented Cu films also have chiral surfaces. The characteristics of the observed LEED patterns, where splitting of diffraction spots is considered to arise from the kinked step features of naturally chiral fcc metal surfaces, are discussed with respect to existing models. These results indicate that the chiral SrTiO₃(621) ceramic surface drives the growth of single-enantiomer, chiral, metal (621) thin films.     

Morphology and formation mechanism of ceria nanoparticles by spray pyrolysis

Ceria-based materials are used in industrial applications such as catalyst supports, carbon monoxide reduction catalysts, and solid oxide fuel cell electrolytes. Various applications require different morphological particles. The ceria particles with various morphologies from the precursors of cerium(III) acetate hydrate, cerium(IV) nitrate hydrate, and cerium(IV) ammonium nitrate were prepared by spray pyrolysis (SP) because SP has the potential for simple and continuous process. The precursor behaviors and the particle morphologies were characterized by thermogravimetric analysis and by transmission electron microscopy. Four main morphologies of solid spherical, hollow spherical with a single pore, hollow concave, and hollow spherical with multiple pores were observed. The experimental results suggest that the morphological formation mechanism is highly correlated with the factors of precursor solubilities, solvent evaporation rates (droplet diameters), and precursor melting temperatures. In addition, total concentrations of cerium(III) in the ceria particles from various precursors were examined using X-ray photoelectron spectroscopy.     

Atomic force microscopy observation of peroxynitrite-induced erythrocyte cytoskeleton reorganization

Atomic force microscopy (AFM) was used to study surface layers of fixed intact erythrocytes. Advantages of simultaneous analysis of surface topography and lateral force maps in the investigation of cytoskeleton structure were shown. Fractal analysis was applied to the lateral force maps of erythrocyte surfaces to evaluate the complexity of the cytoskeleton. Peroxynitrite was used as an oxidant to induce changes in the cytoskeleton structure of intact erythrocytes. Peroxynitrite action on whole blood leads to local abnormalities in the erythrocyte cytoskeleton structure, as well as cytoskeleton reorganization in protruded regions of crenated erythrocytes.     

AFM and contact angle investigation of growth and structure of pp-HMDSO thin films

HMDSO was plasma polymerized on silicon wafer and polyethylene (PE) substrates. The chemical structure of the pp-HMDSO was analyzed with Fourier-transform infrared (FT-IR) spectroscopy. The morphological structure of the thin films deposited on the different substrates was investigated by means of atomic force microscopy (AFM), indicating different coverage mechanisms. In order to investigate the growth process of the pp-HMDSO, films of different thickness were also deposited, varying the plasma deposition time from 10 s to 1800 s. Thickness and structure of such deposits was detected with AFM. Finally, hydrophobic characteristics of the different samples were evaluated by means of contact angle measurements and correlated with the morphological characteristics.