Mechanical properties of epithelial cells in domes investigated using atomic force microscopy
K. Shigemura, K. Kuribayashi-Shigetomi, R. Tanaka, H. Yamasaki, T. Okajima
Frontiers in Cell and Developmental Biology, 11, 1245296, 2023年10月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Atomic force microscopy for investigating cell and tissue mechanics as heterogeneous and hierarchical materials, Journal of Biomechanical Science and Engineering
T. Okajima, K. Kuribayashi-Shigetomi
Journal of Biomechanical Science and Engineering, 2023年10月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Pathogenic Roles of Cardiac Fibroblasts in Pediatric Dilated Cardiomyopathy.
Hirofumi Tsuru, Chika Yoshihara, Hidehiro Suginobe, Mizuki Matsumoto, Yoichiro Ishii, Jun Narita, Ryo Ishii, Renjie Wang, Atsuko Ueyama, Kazutoshi Ueda, Masaki Hirose, Kazuhisa Hashimoto, Hiroki Nagano, Ryosuke Tanaka, Takaharu Okajima, Keiichi Ozono, Hidekazu Ishida
Journal of the American Heart Association, e029676, 2023年06月22日, ［国際誌］
英語, 研究論文（学術雑誌）, Background Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. Methods and Results Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, apoptosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several humoral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor-β signaling pathways were significantly affected in DCM CFs. In contrast, single-cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. Conclusions Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell-cell contact.

Atomic force microscopy identifies the alteration of rheological properties of the cardiac fibroblasts in idiopathic restrictive cardiomyopathy.
Mizuki Matsumoto, Hirofumi Tsuru, Hidehiro Suginobe, Jun Narita, Ryo Ishii, Masaki Hirose, Kazuhisa Hashimoto, Renjie Wang, Chika Yoshihara, Atsuko Ueyama, Ryosuke Tanaka, Keiichi Ozono, Takaharu Okajima, Hidekazu Ishida
PLOS ONE, 17, 9, e0275296, 2022年09月, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Restrictive cardiomyopathy (RCM) is a rare disease characterized by increased ventricular stiffness and preserved ventricular contraction. Various sarcomere gene variants are known to cause RCM; however, more than a half of patients do not harbor such pathogenic variants. We recently demonstrated that cardiac fibroblasts (CFs) play important roles in inhibiting the diastolic function of cardiomyocytes via humoral factors and direct cell-cell contact regardless of sarcomere gene mutations. However, the mechanical properties of CFs that are crucial for intercellular communication and the cardiomyocyte microenvironment remain less understood. In this study, we evaluated the rheological properties of CFs derived from pediatric patients with RCM and healthy control CFs via atomic force microscopy. Then, we estimated the cellular modulus scale factor related to the cell stiffness, fluidity, and Newtonian viscosity of single cells based on the single power-law rheology model and analyzed the comprehensive gene expression profiles via RNA-sequencing. RCM-derived CFs showed significantly higher stiffness and viscosity and lower fluidity compared to healthy control CFs. Furthermore, RNA-sequencing revealed that the signaling pathways associated with cytoskeleton elements were affected in RCM CFs; specifically, cytoskeletal actin-associated genes (ACTN1, ACTA2, and PALLD) were highly expressed in RCM CFs, whereas several tubulin genes (TUBB3, TUBB, TUBA1C, and TUBA1B) were down-regulated. These results implies that the signaling pathways associated with cytoskeletal elements alter the rheological properties of RCM CFs, particularly those related to CF-cardiomyocyte interactions, thereby leading to diastolic cardiac dysfunction in RCM.

Pharmacological Alteration of Cellular Mechanical Properties in Pulmonary Arterial Smooth Muscle Cells of Idiopathic Pulmonary Arterial Hypertension
Shinichi Katsuragi, Nao Tatsumi, Mizuki Matsumoto, Jun Narita, Ryo Ishii, Hidehiro Suginobe, Hirofumi Tsuru, Renjie Wang, Shigetoyo Kogaki, Ryosuke Tanaka, Keiichi Ozono, Takaharu Okajima, Hidekazu Ishida
Cardiology Research, 12, 4, 231, 237, Elmer Press, Inc., 2021年08月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Background: Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease caused by vascular remodeling of the pulmonary arteries with elevated pulmonary vascular resistance. Recently, various pulmonary vasodilator drugs have become available in the clinical field, and have dramatically ameliorated the prognosis of IPAH. However, little is known about how the mechanical properties of pulmonary arterial smooth muscle cells (PASMCs) are altered under drug supplementation. Methods: Atomic force microscopy (AFM) was used to investigate the mechanical properties of PASMCs derived from a patient with IPAH (PAH-PASMCs) and a healthy control (N-PASMCs) which received the supplementation of clinically used drugs for IPAH: sildenafil, macitentan, and riociguat. Results: PASMCs derived from PAH-PASMCs were stiffer than those derived from N-PASMCs. With sildenafil treatment, the apparent Young's modulus (E 0) of cells significantly decreased in PAH-PASMCs but remained unchanged in N-PASMCs. The decrease in E 0 of PAH-PASMCs was also observed in macitentan and riociguat treatment. The stress relaxation AFM revealed that the decrease in E 0 of PAH-PASMCs resulted from a decrease in the cell elastic modulus and/or increase in cell fluidity. The combination treatment of macitentan and riociguat showed an additive effect on cell mechanical properties, implying that this clinically accepted combination therapy for IPAH influences the intracellular mechanical components. Conclusions: Pulmonary vasodilator drugs affect the mechanical properties of PAH-PASMCs, and there exists a mechanical effect of combination treatment on PAH-PASMCs.

Spatiotemporal dynamics of single cell stiffness in the early developing ascidian chordate embryo
Y. Fujii, W. C. Koizumi, T. Imai, M. Yokobori, T. Matsuo, K. Oka, K. Hotta, T. Okajima
Communications Biology, 4, 1, 341, 341, Springer Science and Business Media LLC, 2021年, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.

Measuring viscoelasticity of soft biological samples using atomic force microscopy　　　　　　　　　　　　　　　
Yuri M. Efremov, Takaharu Okajima, Arvind Raman
Soft Matter, 16, 64, 81, 2020年, ［査読有り］
英語, 研究論文（学術雑誌）

Stiffness of brewers’ yeast under ethanol stress investigated by atomic force microscopy　　　　　　　　　　　　　　　
K. Toyota, R. Tanaka, T. Okajima
Japanese Journal of Applied Physics, 59, SN1005, 2020年, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Relationship between rheological properties and actin filaments of single cells investigated by atomic force microscopy　　　　　　　　　　　　　　　
R. Tanaka, M. Sawano, Y. Fujii, K. Kuribayashi-Shigetomi, A. Subagyo, K. Sueoka, T. Okajima*
Japanese Journal of Applied Physics, 59, SN1010, 2020年, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Spontaneous Spatial Correlation of Elastic Modulus in Jammed Epithelial Monolayers Observed by AFM.
Yuki Fujii, Yuki Ochi, Masahiro Tuchiya, Mihoko Kajita, Yasuyuki Fujita, Yukitaka Ishimoto, Takaharu Okajima
Biophysical journal, 116, 6, 1152, 1158, 2019年03月19日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, For isolated single cells on a substrate, the intracellular stiffness, which is often measured as the Young's modulus, E, by atomic force microscopy (AFM), depends on the substrate rigidity. However, little is known about how the E of cells is influenced by the surrounding cells in a cell population system in which cells physically and tightly contact adjacent cells. In this study, we investigated the spatial heterogeneities of E in a jammed epithelial monolayer in which cell migration was highly inhibited, allowing us to precisely measure the spatial distribution of E in large-scale regions by AFM. The AFM measurements showed that E can be characterized using two spatial correlation lengths: the shorter correlation length, lS, is within the single cell size, whereas the longer correlation length, lL, is longer than the distance between adjacent cells and corresponds to the intercellular correlation of E. We found that lL decreased significantly when the actin filaments were disrupted or calcium ions were chelated using chemical treatments, and the decreased lL recovered to the value in the control condition after the treatments were washed out. Moreover, we found that lL decreased significantly when E-cadherin was knocked down. These results indicate that the observed long-range correlation of E is not fixed within the jammed state but inherently arises from the formation of a large-scale actin filament structure via E-cadherin-dependent cell-cell junctions.

Cricket tympanal organ revisited: morphology, development, and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane
H. Nishino, M. Domae, T. Takanashi, T. Okajima
Cell and Tissue Research, 377, 2, 1, 22, Springer Science and Business Media LLC, 2019年, ［査読有り］
英語, 研究論文（学術雑誌）

Calibrating the Young’s modulus of soft materials with surface tilt angle measured by atomic force microscopy　　　　　　　　　　　　　　　
Y. Fujii, T. Okajima
AIP Advances, 9, 015028, 2019年, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Fascin in lamellipodia contributes to cell elasticity by controlling the orientation of filamentous actin
M. Tanaka, Y. Fujii, K. Hirano, T. Higaki, A. Nagasaki, R. Ishikawa, T. Okajima, K. Katoh
Genes to Cells, 24, 3, 202, 213, 2019年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.

Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion
Y. Miyatake, K. Kuribayashi-Shigetomi, Y. Ohta, S. Ikeshita, A. Subagyo, K. Sueoka, A. Kakugo, M. Amano, T. Takahashi, T. Okajima, M. Kasahara
Scientific Reports, 8, 1, 14054, Springer Science and Business Media LLC, 2018年, ［査読有り］
英語, 研究論文（学術雑誌）

Origami-based self-folding of co-cultured NIH/3T3 and HepG2 cells into 3D microstructure
Q. He, T. Okajima, H. Onoe, A. Subagyo, K. Sueoka, K. Kuribayashi-Shigetomi
Scientific Reports, 8, 1, 4556, Springer Science and Business Media LLC, 2018年, ［査読有り］
英語, 研究論文（学術雑誌）

Regulation of Neuritogenesis in Hippocampal Neurons using Stiffness of Extracellular Microenvironment
A. Tanaka, Y. Fujii, N. Kasai, T. Okajima, H. Nakashima
PLOS ONE, 13, 2, e019192, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The mechanosensitivity of neurons in the central nervous system (CNS) is an interesting issue as regards understanding neuronal development and designing compliant materials as neural interfaces between neurons and external devices for treating CNS injuries and disorders. Although neurite initiation from a cell body is known to be the first step towards forming a functional nervous network during development or regeneration, less is known about how the mechanical properties of the extracellular microenvironment affect neuritogenesis. Here, we investigated the filamentous actin (F-actin) cytoskeletal structures of neurons, which are a key factor in neuritogenesis, on gel substrates with a stiffness-controlled substrate, to reveal the relationship between substrate stiffness and neuritogenesis. We found that neuritogenesis was significantly suppressed on a gel substrate with an elastic modulus higher than the stiffness of in vivo brain. Fluorescent images of the F-actin cytoskeletal structures showed that the F-actin organization depended on the substrate stiffness. Circumferential actin meshworks and arcs were formed at the edge of the cell body on the stiff gel substrates unlike with soft substrates. The suppression of F-actin cytoskeleton formation improved neuritogenesis. The results indicate that the organization of neuronal F-actin cytoskeletons is strongly regulated by the mechanical properties of the surrounding environment, and the mechanically-induced F-actin cytoskeletons regulate neuritogenesis.

Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population
PingGen Cai, Ryosuke Takahashi, Kaori Kuribayashi-Shigetomi, Agus Subagyo, Kazuhisa Sueoka, John M. Maloney, Krystyn J. Van Vliet, Takaharu Okajima
Biophysical Journal, 113, 3, 671, 678, CELL PRESS, 2017年08月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G(star)). Although the ensemble variation in G(star) of single cells has been elucidated, the detailed temporal variation of G(star) remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G(star) implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale.

Mechanical properties of paraformaldehyde‑treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy　　　　　　　　　　　　　　　
S.‐O. Kim, J. Kim, T. Okajima, N.‐J. Cho
Nano Convergence, 4, 5, 2017年, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）

Comparison between power-law rheological parameters of living cells in frequency and time domains measured by atomic force microscopy
Ryosuke Takahashi, Takaharu Okajima
JAPANESE JOURNAL OF APPLIED PHYSICS, 55, 8, 08NB22, IOP PUBLISHING LTD, 2016年08月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, We investigated how stress relaxation mapping is quantified compared with the force modulation mapping of confluent epithelial cells using atomic force microscopy (AFM). Using a multi-frequency AFM technique, we estimated the power-law rheological behaviors of cells simultaneously in time and frequency domains. When the power-law exponent alpha was low (< 0.1), the alpha values were almost the same in time and frequency domains. On the other hand, we found that at the high values (alpha > 0.1), alpha in the time domain was underestimated relative to that in the frequency domain, and the difference increased with alpha, whereas the cell modulus was overestimated in the time domain. These results indicate that power-law rheological parameters estimated by stress relaxation are sensitive to lag time during initial indentation, which is inevitable in time-domain AFM experiments. (C) 2016 The Japan Society of Applied Physics.

Simple and rapid formation of 3D co-culture cell laden microstructures by using cell origami technique
Q. He, T. Okajima, K. K. Shigetomi
20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, 3, 4, 2016年
研究論文（国際会議プロシーディングス）, This paper described utilizing cell traction force to form three dimension (3D) co-culture cell laden microstructures simply and rapidly by using cell origami technique (Fig. 1) and the function detection of these 3D co-culture cells. Seeding different kinds of cells and lifted off the microplates, then the stretched cells can immediately fold the microplates by their traction force and formed 3D microstructures. Furthermore, the higher function was also detected in co-culture cells inside of 3D microstructures. We believe these microstructures will be available for investigating many functions by combining with different co-culture cells in various situations.

Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy
Ryosuke Takahashi, Takaharu Okajima
APPLIED PHYSICS LETTERS, 107, 17, AMER INST PHYSICS, 2015年10月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50-500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G*. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods. (C) 2015 AIP Publishing LLC.

Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy
Aya Tanaka, Ryosuke Tanaka, Nahoko Kasai, Shingo Tsukada, Takaharu Okajima, Koji Sumitomo
JOURNAL OF STRUCTURAL BIOLOGY, 191, 1, 32, 38, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes. (C) 2015 Elsevier Inc. All rights reserved.

Precision of cell-to-cell variation in power-law rheology characterized by atomic force microscopy
PingGen Cai, Takaharu Okajima
JAPANESE JOURNAL OF APPLIED PHYSICS, 54, 3, IOP PUBLISHING LTD, 2015年03月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, The mechanical properties of compliant single cells are significantly associated with various cell functions. It is thus crucially important in the identification and sorting of cells to characterize not only the complex moduli that exhibit power-law rheology but also the cell-to-cell variation at the single cell level. Atomic force microscopy (AFM) can be used to measure cellular mechanical properties and to quantify cell-to-cell variation. However, less is known about how precisely and routinely the cell-to-cell variation is obtained when the complex moduli vary substantially in different cell samples. Here, we investigate the storage modulus G' for single cells measured at controlled positions by AFM. We find that the spatial dependence of the frequency-dependent component of the cell-to-cell variation is preserved even if the spatial heterogeneity of G' is changed with the cell sample. The invariance of the frequency-dependent cell-to-cell variation indicates the robustness of AFM for the mechanical diagnosis of single cells. (C) 2015 The Japan Society of Applied Physics

Atomic force microscopy for mapping mechanical property of the whole cell assembly
Ryosuke Tanaka, Yuki Fujii, Junpei Kikkawa, Kaori Kuribayashi-Shigetomi, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
2014 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2014, Institute of Electrical and Electronics Engineers Inc., 2015年01月09日
英語, 研究論文（国際会議プロシーディングス）, Cells rapidly undergo cell division during the embryogenesis. Such dynamic behaviors of cells during the embryogenesis are considered to be strongly associated with their mechanical properties such as cell-cell mechanical interactions and cell stiffness. However, the interplay between the morphogenesis and the mechanical property of whole cell assembly during the developmental process has not been well understood. To exploring the mechanism of forming the whole cell assembly, we proposed atomic force microscopy (AFM) combined with a microarray technique, which allows us to map mechanical property of the whole cell assembly. In this AFM setup, the cell assembly is randomly directed in the microarray well, and thus the average mechanical property of the whole cell assembly can be reconstructed from mapping images obtained from different cell assemblies.

Atomic Force Microscopy: Imaging and Rheology of Living Cells　　　　　　　　　　　　　　　
Takaharu Okajima
Nano/Micro Science and Technology in Biorheology, 2015年, ［招待有り］

High-throughput Measurements of Single Cell Rheology by Atomic Force Microscopy　　　　　　　　　　　　　　　
High-throughput Measurements of Single Cell Rheology by, Atomic Force Microscopy, Kaori Kuribayashi-Shigetomi, Ryosuke Takahashi, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
Hyper Bio Assembler for 3D Cellular Systems, 57, 67, 2015年, ［最終著者, 責任著者］
英語

Filamin acts as a key regulator in epithelial defence against transformed cells
Mihoko Kajita, Kaoru Sugimura, Atsuko Ohoka, Jemima Burden, Hitomi Suganuma, Masaya Ikegawa, Takashi Shimada, Tetsuya Kitamura, Masanobu Shindoh, Susumu Ishikawa, Sayaka Yamamoto, Sayaka Saitoh, Yuta Yako, Ryosuke Takahashi, Takaharu Okajima, Junichi Kikuta, Yumiko Maijima, Masaru Ishii, Masazumi Tada, Yasuyuki Fujita
NATURE COMMUNICATIONS, 5, NATURE PUBLISHING GROUP, 2014年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Recent studies have shown that certain types of transformed cells are extruded from an epithelial monolayer. However, it is not known whether and how neighbouring normal cells play an active role in this process. In this study, we demonstrate that filamin A and vimentin accumulate in normal cells specifically at the interface with Src- or RasV12-transformed cells. Knockdown of filamin A or vimentin in normal cells profoundly suppresses apical extrusion of the neighbouring transformed cells. In addition, we show in zebrafish embryos that filamin plays a positive role in the elimination of the transformed cells. Furthermore, the Rho/Rho kinase pathway regulates filamin accumulation and filamin acts upstream of vimentin in the apical extrusion. This is the first report demonstrating that normal epithelial cells recognize and actively eliminate neighbouring transformed cells and that filamin is a key mediator in the interaction between normal and transformed epithelial cells.

Atomic force microscopy measurements of mechanical properties of single cells patterned by microcontact printing
Ryosuke Takahashi, Satoshi Ichikawa, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
ADVANCED ROBOTICS, 28, 7, 449, 455, TAYLOR & FRANCIS LTD, 2014年04月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, To investigate the mechanical properties of adherent cells under conditions where cell-to-cell interaction can be prevented and controlled, we used microcontact printing to pattern single cells without intercellular contacts and measured them with a custom-built atomic force microscopy (AFM) system. The motorized AFM system can measure individual cells over a large spatial range, enabling the measurement of cells in a microarray format. We tested single fibroblast cells and found that the power-law of their complex shear modulus is not significantly influenced by cell-to-cell contact. This method is effective for obtaining high-throughput measurements on single cells.

Temporal Change in Complex Shear Modulus of Cells: An Atomic Force Microscopy Study
PingGen Cai, Ryosuke Takahashi, Kaori Kuribayashi-Shigetomi, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
2014 INTERNATIONAL SYMPOSIUM ON MICRO-NANOMECHATRONICS AND HUMAN SCIENCE (MHS), 1, 3, IEEE, 2014年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, To sort living cells according to our needs, it is important to understand how degree cell property measured for cell sorting fluctuates in time. Mechanical property of cells is one of essential indicators for cell sorting. Thus, in this study, we attempted to measure a time evolution of viscoelastic property such as complex shear modulus, G* of single cells adhered on substrates using atomic force microscopy (AFM). We observed that the G* largely fluctuated in time even the cells are placed on substrates in a confined condition. This indicates that in mechanical cell sorting, mechanical fluctuations of cells should be carefully estimated so that cells are precisely separated by taking the measured data involving cell fluctuations into account.

Quantitative Rheological Measurements of Confluent Cell Using Atomic Force Microscopy
Ryosuke Takahashi, Kaori Kuribayashi-Shigetomi, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
2014 INTERNATIONAL SYMPOSIUM ON MICRO-NANOMECHATRONICS AND HUMAN SCIENCE (MHS), 1, 3, IEEE, 2014年, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）, Rheological properties of cells are associated with various cell functions and thus are considered to be an indicator for diagnosing cell disease. Atomic force microscopy (AFM) is a powerful tool for quantifying the mechanical properties of isolated single cells. For example, our AFM technique reported previously revealed that the complex shear modulus of single cells exhibited a large cell-to-cell variation which depended on frequency. By contrast, rheological properties of cell population such as cells in confluent condition have been less understood. Thus, it is valuable to investigate how quantitatively the AFM technique can be applied to cell population such as cells in confluent condition. As a result, rheological properties of cells in confluent conditions can be relatively rapidly measured by our AFM setup modifying a force modulation AFM mode. This suggests that the AFM technique is useful for diagnosing not only single cells but also cell population and cell assembly.

Quantifying cell-to-cell variation in power-law rheology
Pinggen Cai, Yusuke Mizutani, Masahiro Tsuchiya, John M. Maloney, Ben Fabry, Krystyn J. Van Vliet, Takaharu Okajima
Biophysical Journal, 105, 5, 1093, 1102, 2013年09月03日, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Among individual cells of the same source and type, the complex shear modulus exhibits a large log-normal distribution that is the result of spatial, temporal, and intrinsic variations. Such large distributions complicate the statistical evaluation of pharmacological treatments and the comparison of different cell states. However, little is known about the characteristic features of cell-to-cell variation. In this study, we investigated how this variation depends on the spatial location within the cell and on the actin filament cytoskeleton, the organization of which strongly influences cell mechanics. By mechanically probing fibroblasts arranged on a microarray, via atomic force microscopy, we observed that the standard deviation σ of was significantly reduced among cells in which actin filaments were depolymerized. The parameter σ also exhibited a subcellular spatial dependence. Based on our findings regarding the frequency dependence of σ of the storage modulus G′, we proposed two types of cell-to-cell variation in G′ that arise from the purely elastic and the frequency-dependent components in terms of the soft glassy rheology model of cell deformability. We concluded that the latter inherent cell-to-cell variation can be reduced greatly by disrupting actin networks, by probing at locations within the cell nucleus boundaries distant from the cell center, and by measuring at high loading frequencies. © 2013 Biophysical Society.

Nanoscale fluctuations on epithelial cell surfaces investigated by scanning ion conductance microscopy
Yusuke Mizutani, Myung-Hoon Choi, Sang-Joon Cho, Takaharu Okajima
APPLIED PHYSICS LETTERS, 102, 17, AMER INST PHYSICS, 2013年04月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Nanoscale fluctuations on the apical surfaces of epithelial cells connected to neighboring cells were investigated by scanning ion conductance microscopy. Mapping the ion current as a function of the tip-surface distance revealed that in untreated cells, the apparent fluctuation amplitude increased towards the cell center. We found that the spatial dependence was less correlated with the heterogeneities of cell stiffness but was significantly reduced when actin filaments were disrupted. The results indicate that apical surface fluctuations are highly constrained at the cell-cell interface, in the vertical direction to the surface and by the underlying actin filaments. (C) 2013 AIP Publishing LLC.

High-throughput measurements of cell mechanics using atomic force microscopy with micro-patterned substrates.
Ryosuke Takahashi, Kaori Kuribayashi-Shigetomi, Masahiro Tsuchiya, Agus Subagyo, Kazuhisa Sueoka, Takaharu Okajima
International Symposium on Micro-NanoMechatronics and Human Science, MHS 2013, Nagoya, Japan, November 10-13, 2013, 1, 3, IEEE, 2013年, ［査読有り］
研究論文（国際会議プロシーディングス）

原子間力顕微鏡を用いた細胞形状を制御した単一細胞のレオロジー空間測定
市川 論, 水谷 祐輔, 高橋 亮輔, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2012.1, 2550, 2550, 公益社団法人 応用物理学会, 2012年02月29日
日本語

イオンコンダクタンス顕微鏡による細胞膜揺らぎ測定
水谷 祐輔, 蔡 萍根, 土屋 雅博, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2012.1, 1595, 1595, 公益社団法人 応用物理学会, 2012年02月29日
日本語

Effect of isoproterenol on local contractile behaviors of rat cardiomyocytes measured by atomic force microscopy
Yusuke Mizutani, Koichi Kawahara, Takaharu Okajima
Current Pharmaceutical Biotechnology, 13, 14, 2599, 2603, Bentham Science Publishers B.V., 2012年, ［査読有り］
英語, 研究論文（学術雑誌）, Inotropic agents induce changes in the contraction amplitude and frequency of cardiomyocytes (CMs). However, it is unknown how local contractions of CMs treated by inotropic agents behave spatiotemporally. In this study, the effect of isoproterenol, a positive inotropic agent, on local contractions of isolated neonatal rat CMs was explored by atomic force microscopy (AFM). We observed that changes in local contraction amplitude of CM in the presence of isoproterenol were heterogeneous
they were unchanged or increased, at different positions, with respect to the amplitude of untreated CMs. Interestingly, spatial heterogeneities of local contraction amplitude of CM in the presence of isoproterenol did not obviously correlate with the local elasticity, indicating that the local contractions were facilitated by cooperative dynamics of the cytoskeletal structure in relatively large regions, rather than those just under AFM indentation. Moreover, local contraction amplitude of CM in the presence of isoproterenol was not proportional to that in the control condition, showing that the former change was no longer additive in local scales. © 2012 Bentham Science Publishers.

Atomic force microscopy for the examination of single cell rheology
Takaharu Okajima
Current Pharmaceutical Biotechnology, 13, 14, 2623, 2631, Bentham Science Publishers B.V., 2012年, ［査読有り］
英語, Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM. © 2012 Bentham Science Publishers.

Direct observation of dynamic force propagation between focal adhesions of cells on microposts by atomic force microscopy
Akinori Okada, Yusuke Mizutani, Agus Subagyo, Hirotaka Hosoi, Motonori Nakamura, Kazuhisa Sueoka, Koichi Kawahara, Takaharu Okajima
APPLIED PHYSICS LETTERS, 99, 26, 263703, AMER INST PHYSICS, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, We investigated dynamic force propagation between focal adhesions of fibroblast cells cultured on polydimethylsiloxane micropost substrates, by atomic force microscopy. Live cells were mechanically modulated by the atomic force microscopy probe bound to cell apical surfaces at 0.01-0.5 Hz, while microposts served as a force sensor at basal surfaces. We observed that cells exhibited rheological behavior at the apical surface but had no apparent out-of-phase response at the basal surface, indicating that the dynamic force propagating through cytoskeletal filaments behaves in an elastic manner. Moreover, the direction of the propagated force was observed to be intimately associated with the prestress. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3672225]

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia
Kunugi, Satohiko, Iwabuchi, Sadahiro, Matsuyama, Daisuke, Okajima, Takaharu, Kawahara, Koichi
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 416, 3-4, 409, 415, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. lschemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2 h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes. (C) 2011 Elsevier Inc. All rights reserved.

AFMによる単一細胞レオロジーの個体差の精密解析
蔡 萍根, 水谷 祐輔, 土屋 雅博, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2011.2, 1077, 1077, 公益社団法人 応用物理学会, 2011年08月16日
日本語

Rheological Properties of Growth-Arrested Fibroblast Cells under Serum Starvation Measured by Atomic Force Microscopy
Atsushi Miyaoka, Yusuke Mizutani, Masahiro Tsuchiya, Koichi Kawahara, Takaharu Okajima
JAPANESE JOURNAL OF APPLIED PHYSICS, 50, 8, 08LB16, IOP PUBLISHING LTD, 2011年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The rheological properties of growth-arrested and quiescent (G0 phase) mouse fibroblast cells under serum starvation were investigated by atomic force microscopy (AFM) with a microarray technique. The number distribution of complex shear modulus, G*, of quiescent cells at the serum concentration, C-S = 0.1%, followed a log-normal distribution, and the frequency dependence of G* exhibited a power law behavior, which were similar to those under a control condition at CS 10%. On the other hand, we found that the Newtonian viscosity coefficient of the quiescent cells significantly increased, and the distribution broadened, as compared with the control cells, whereas the power-law exponent was unchanged. The result indicated that the rheological properties of quiescent fibroblast cells were not identical to those in the G1 phase during cell cycle. This finding suggests that the Newtonian viscosity of cells is one of the useful indicators for evaluating growth-arrested cells under serum starvation. (C) 2011 The Japan Society of Applied Physics

AFMによる細胞内ネットワーク構造変化に対する細胞粘弾性の研究
蔡 萍根, 水谷 祐輔, 土屋 雅博, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2011.1, 1637, 1637, 公益社団法人 応用物理学会, 2011年03月09日
日本語

AFMによる不死化過程における細胞粘弾性の細胞数分布解析
水谷 祐輔, 蔡 萍根, 土屋 雅博, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2011.1, 1636, 1636, 公益社団法人 応用物理学会, 2011年03月09日
日本語

原子間力顕微鏡を用いた物理刺激による細胞カルシウム応答測定
佐藤 淳平, 水谷 祐輔, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2011.1, 2723, 2723, 公益社団法人 応用物理学会, 2011年03月09日
日本語

Artificial polymeric receptors on the cell surface promote the efficient cellular uptake of quantum dots
Kenichi Niikura, Katsuyuki Nambara, Takaharu Okajima, Ryosuke Kamitani, Shin Aoki, Yasutaka Matsuo, Kuniharu Ijiro
ORGANIC & BIOMOLECULAR CHEMISTRY, 9, 16, 5787, 5792, ROYAL SOC CHEMISTRY, 2011年, ［査読有り］
英語, 研究論文（学術雑誌）, In our previous paper, secondary-amine appended cationic polymer 1 was used as a scaffold to display artificial receptors on a cell surface (R. Kamitani et al., ChemBioChem, 2009, 10, 230). This polymer can be retained on the cell surface for more than 30 min before being slowly internalized into the cells. In this study, our aim is to achieve the efficient internalization of quantum dots (QDs) into target cells via artificial receptors on the polymer. As a receptor molecule, N-acetylglucosamine (GlcNAc) moieties were introduced into the polymer, and GlcNAc binding protein-displaying QDs were used as a ligand. We found that ligand-presenting QDs could be internalized effectively into cells via polymer-mediated endocytosis, whereas QDs were not internalized into untreated cells. These data suggest that our method based on cell-surface engineering using polymers affords a new approach to the delivery of various poorly permeable nanoparticles into cells.

Influence of Hydrophobic Structures on the Plasma Membrane Permeability of Lipidlike Molecules
Kenichi Niikura, Katsuyuki Nambara, Takaharu Okajima, Yasutaka Matsuo, Kuniharu Ijiro
LANGMUIR, 26, 12, 9170, 9175, AMER CHEMICAL SOC, 2010年06月, ［査読有り］
英語, 研究論文（学術雑誌）, A series of FITC-labeled hydrophobic molecules (1-8) were prepared, and their cellular uptakes have been investigated using cell-cycle-synchronized HeLa cells. The cellular membrane permeability of compounds strongly depended on both the chemical structure and the cell-cycle phase. In the G1/S phase, branched hydrocarbon-containing 3 and cis-olefin-containing 2 and 8 were efficiently internalized into cells by passive diffusion. In contrast, linear alkyl chain-containing 1 and 7 were retained on the membrane without rapid internalization. In the M phase, rapid permeation was suppressed for all molecules.

AFMによる形質転換過程における線維芽細胞の複素弾性率の解析
水谷 祐輔, 蔡 萍根, 宮岡 敦史, 土屋 雅博, 春菜 ゆかり, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2010.1, 2674, 2674, 公益社団法人 応用物理学会, 2010年03月03日
日本語

AFM による多数細胞レオロジーの普遍性と細胞個性の評価
蔡 萍根, 水谷 祐輔, 宮岡 敦史, 土屋 雅博, 河原 剛一, 岡嶋 孝治
応用物理学会学術講演会講演予稿集, 2010.1, 1526, 1526, 公益社団法人 応用物理学会, 2010年03月03日
日本語

Statistics of Single Cell Mechanics Investigated by Atomic Force Microscopy　　　　　　　　　　　　　　　
Hiratsuka, Y. Mizutani, P.G. Cai, M. Tsuchiya, H. Tokumoto, K. Kawahara, T. Okajima
MRS Spring Meeting Symposium U proceedings, 9999, 2010年, ［査読有り］

Power-Law Stress and Creep Relaxations of Single Cells Measured by Colloidal Probe Atomic Force Microscopy
Shinichiro Hiratsuka, Yusuke Mizutani, Akitoshi Toda, Norichika Fukushima, Koichi Kawahara, Hiroshi Tokumoto, Takaharu Okajima
JAPANESE JOURNAL OF APPLIED PHYSICS, 48, 8, 08JB14-4, IOP PUBLISHING LTD, 2009年08月, ［査読有り］
英語, 研究論文（学術雑誌）, We measured stress and creep relaxations of mouse fibroblast cells arranged and cultured on a microarray, by colloidal probe atomic force microscopy (AFM). A hydrophobic monolayer coating of perfluorodecyltrichlorosilane (FDTS) on the surface of colloidal silica beads significantly reduced the adhesion force of live cells, compared with untreated beads. The rheological behaviors of cells were estimated by averaging several relaxation curves of cells measured by the AFM. Longer-time tailing of both stress and creep relaxation curves followed single power-law behavior over a time scale of 60s, with exponents in the range 0.1-0.4, varying with cells. The results were in good agreement with previous measurements of the frequency-domain rheology of cells using the force modulation mode. (C) 2009 The Japan Society of Applied Physics

The number distribution of complex shear modulus of single cells measured by atomic force microscopy
Shinichiro Hiratsuka, Yusuke Mizutani, Masahiro Tsuchiya, Koichi Kawahara, Hiroshi Tokumoto, Takaharu Okajima
ULTRAMICROSCOPY, 109, 8, 937, 941, ELSEVIER SCIENCE BV, 2009年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The viscoelastic properties of a large number of mouse fibroblast NIH3T3 cells (n similar or equal to 130) were investigated by combining atomic force microscopy (AFM) with a microarray technique. In the experiments, the cells were arranged and cultured in the wells of a microarray substrate, and a force modulation mode experiment was used to measure the complex shear modulus, G*, of individual cells in a frequency range 0.5-200 Hz. The frequency dependence of G* of the cells exhibited a power-law behavior and similar frequency dependencies have been observed in several cell types cultured on flat substrates. This indicated that the NIH3T3 cells cultured in the wells of a microarray have analogous structural organization to those cells cultured on flat substrates. The number distribution of both the storage and loss moduli of G* fitted well to a log-normal distribution function, whereas the power-law exponent estimated by a power-law structural damping model showed a normal distribution function. These results showed that combining AFM with a microarray technique was a suitable approach for investigating the statistics of rheological properties of living cells without the requirement of cell surface modification. (c) 2009 Elsevier B.V. All rights reserved.

Design of Cell-Surface-Retained Polymers for Artificial Ligand Display
Ryosuke Kamitani, Kenichi Niikura, Takaharu Okajima, Yasutaka Matsuo, Kuniharu Ijiro
CHEMBIOCHEM, 10, 2, 230, 233, WILEY-V C H VERLAG GMBH, 2009年01月, ［査読有り］
英語, 研究論文（学術雑誌）

Localized yielding around crack tips of double-network gels(a)
Yoshimi Tanaka, Yasunori Kawauchi, Takayuki Kurokawa, Hidemitsu Furukawa, Takaharu Okajima, Jian Ping Gong
MACROMOLECULAR RAPID COMMUNICATIONS, 29, 18, 1514, 1520, WILEY-V C H VERLAG GMBH, 2008年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Double-network (DN) gels, a type of interpenetrating polymer network (IPN) consisting of rigid and flexible polymer components, exhibit two outstanding mechanical behaviors: yielding deformation of the entire specimen in tensile tests and quite high fracture energy in tearing tests. In this study, atomic force microscope (AFM) measurements were conducted on DN gels to determine the local Young's moduli immediately below the fracture surfaces E-f and below the usual molded surfaces E-m, and compare the local modulus with bulk Young's moduli measured before and after the yielding deformation, denoted as E-h and E-s, respectively. E-m and E-h are around 0.1 MPa; E-f and E-s, around 0.01 MPa, one order lower than the former two moduli. The order relation indicates that yielding deformation occurred locally around the crack tip of the DN gel during fracture. This supports the basic assumption of phenomenological models recently proposed to explain high fracture energy of DN gels.

Elasticity of living cells on a microarray during the early stages of adhesion measured by atomic force microscopy
Yusuke Mizutani, Masahiro Tsuchiya, Shinichiro Hiratsuka, Koichi Kawahara, Hiroshi Tokumot, Takaharu Okajima
JAPANESE JOURNAL OF APPLIED PHYSICS, 47, 7, 6177, 6180, JAPAN SOCIETY APPLIED PHYSICS, 2008年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The number distribution of the elastic modulus of fibroblast Cells Was successfully measured during the early stages of adhesion using an atomic force microscope (AFM) combined with a microarray as a substrate, which allowed us to arrange and Culture cells so that a large number of cells could be measured in a short time period. We confirmed that the cells deposited in the wells of the microarray could be cultured for at least 12 h without any significant migration. Histograms of the Young's modulus. E. of the cells during the early stages of adhesion produced from force curve measurements of cells (n congruent to 300) cultured for 3-9 h were well fitted to a log-normal distribution function. With increasing incubation time, the average value of E increased significantly, while the standard deviation of the distribution remained almost constant. The results are discussed in terms of the cytoskeleton inside cells.

Affinity of functional groups for membrane surfaces: Implications for physically irreversible fouling
Hiroshi Yamamura, Katsuki Kimura, Takaharu Okajima, Hiroshi Tokumoto, Yoshimasa Watanabe
ENVIRONMENTAL SCIENCE & TECHNOLOGY, 42, 14, 5310, 5315, AMER CHEMICAL SOC, 2008年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Fouling in membranes used for water treatment has been attributed to the presence of natural organic matter (NOM) in water. There have been reports recently on the contribution of hydrophilic fractions of NOM(e.g., carbohydrate-like substances) to fouling, but there is still little information about the physicochemical interactions between membranes and carbohydrate-like substances. In this study, the affinity of carbohydrate-like substances to two different microfiltration (MF) membranes was investigated by using atomic force microscopy (AFM) and functionally modified microspheres. Microspheres were attached to the tip of the cantilever in an AFM apparatus and the adhesion forces working between the microspheres and the membranes were determined. The microspheres used in this study were coated with either hydroxyl groups or carboxyl groups to be used as surrogates of carbohydrate-like substances or humic acid, respectively. Measurements of adhesion force were carried out at pH of 6.8 and the experimental results demonstrated that the adhesion force to membranes was strong in the case of hydroxyl groups but weak in the case of carboxyl groups. The strong adhesion between the hydroxyl group and the membrane surface is explained by the strong hydrogen bond generated. It was also found that the affinity of the hydroxyl group to a polyvinylidenefluoride (PVDF) membrane was much higher than that to a polyethylene (PE) membrane, possibly due to the high electronegative nature of the PVDF polymer. The time course of changes in the affinity of hydroxyl group to a membrane used in a practical condition was investigated by repeatedly carrying out AFM force measurements with PE membrane specimens sampled from a pilot plant operated at an existing water treatment plant. Microspheres exhibited strong affinity to the membrane at the initial stage of operation (within 5 days), but subsequently exponential reduction of the affinity was seen until the end of operation, as a result of fouling development. However, the magnitude of affinity of hydroxyl-modified microspheres was much higher than that of carboxyl-modified microspheres even after the significant reduction of affinity of hydroxyl-modified microspheres to the membranes was seen. The results obtained in this study partially explain why hydrophilic NOM dominated over humic substances in foulants of membranes used for water treatment in recent studies on fouling.

Observation of the three-dimensional structure of actin bundles formed with polycations
Kazuhiro Shikinaka, Hyuckjoon Kwon, Akira Kakugo, Hidemitsu Furukawa, Yoshihito Sada, Jian Ping Gong, Yoshitaka Aoyama, Hideo Nishioka, Hiroshi Jinnai, Takaharu Okajima
BIOMACROMOLECULES, 9, 2, 537, 542, AMER CHEMICAL SOC, 2008年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Three-dimensional structures of actin bundles formed with polycations were observed by using transmission electron microtomography and atomic force microscopy. We found, for the first time, that the cross-sectional morphology of actin bundles depends on the polycation species and ionic strength, while it is insensitive to the degree of polymerization and concentration of polycation. Actin bundles formed with poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary show a ribbon-like cross-sectional morphology in low salt concentrations that changes to cylindrical cross-sectional morphology with hexagonal packing of the actin filaments in high salt concentrations. Contrastingly, actin bundles formed with poly-L-lysine show triangular cross-sectional morphology with hexagonal packing of the actin filaments. These variations in cross-sectional morphology are discussed in terms of anisotropy in the electrostatic energy barrier.

AFMによる多数細胞の表面力学測定
水谷 祐輔, 平塚 伸一郎, 土屋 雅博, 河原 剛一, 徳本 洋志, 岡嶋 孝治
表面科学学術講演会要旨集, 28, 185, 185, 公益社団法人 日本表面真空学会, 2008年
日本語, 原子間力顕微鏡（AFM）による細胞表面力学計測は、現在まで多数の研究がなされおり，１個または数個の細胞を用いた力学計測がなされてきた。一方で、細胞力学をより詳細に定量化するためには、多数の細胞を測定し，統計的な評価をすることが望まれる．本研究では，マイクロアレイ技術をAFM測定に利用して，多数の細胞の力学特性を高速に計測する方法を開発し、それを用いた細胞力学測定を行った．

Nanorheology of living cells investigated by atomic force microscopy　　　　　　　　　　　　　　　
T. Okajima, H. Tokumoto
Journal of the society of rheology, 36, 81, 86, 2008年, ［筆頭著者］
日本語

Stress relaxation measurement of fibroblast cells with atomic force microscopy
Takaharu Okajima, Masaru Tanaka, Shusaku Tsukiyama, Tsubasa Kadowaki, Sadaaki Yamamoto, Masatsugu Shimomura, Hiroshi Tokumot
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS, 46, 8B, 5552, 5555, INST PURE APPLIED PHYSICS, 2007年08月, ［査読有り］
英語, 研究論文（学術雑誌）, We measured the stress relaxation of mouse fibroblast NIH3T3 cells with an atomic force microscope (AFM) using a sharp silicon tip and a silica bead with a radius of similar to 1 mu m as an indenter. The decay of loading force was clearly observed in NIH3T3 cells at a small initial loading force of similar to 0.4 nN and was well fitted to the stretched exponential function rather than to a single exponential function. The stretching exponent parameter was similar to 0.5 for both indenters, indicating that the stress relaxation observed in NIH3T3 cells consisted of multiple relaxation processes. The time-domain AFM technique described in this report allows us to measure directly the relaxation process of living cells in a range from milliseconds to seconds.

Actin network formation by unidirectional polycation diffusion
Hyuck Joon Kwon, Akira Kakugo, Takehiro Ura, Takaharu Okajima, Yoshimi Tanaka, Hidemitsu Furukawa, Yoshihito Osada, Jian Ping Gong
LANGMUIR, 23, 11, 6257, 6262, AMER CHEMICAL SOC, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, We show that F-actins form three-dimensional giant network under uni-directional diffusion of polycations, at a dilute actin concentration (0.01 mg/mL) that only bundles are formed by homogeneous mixing with polycations. The mesh size of the actin network depends on polycation concentration and ionic strength, while bundle thickness of network depends only on ionic strength, which indicates that actin network is formed through nucleation-growth mechanism. The mesh size and the bundle thickness are determined by nucleus concentration and nucleus size, respectively. The atomic force microscopy measurement correlates the elasticity of the actin network, E, with the mesh size, xi, as E similar to xi(-1), while the bundle thickness, D dependence of E cannot be described by a simple scaling relation. E similar to D-6.5 when D is small and E similar to D-0.1 when D is large. Our study on the self-assembly of actin network under asymmetric polycation condition would provide the crucial insight into the organization of biopolymers in polarized condition of cell.

Force spectroscopy on single polymer incorporated into polymer gels
Takaharu Okajima, Xiang-Ming Tao, Hiroaki Azehara, Hiroshi Tokumoto
JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY, 7, 3, 790, 795, AMER SCIENTIFIC PUBLISHERS, 2007年03月, ［査読有り］
英語, 研究論文（学術雑誌）, We conducted the extraction experiments of single polymer incorporated into hydrogels with an atomic force microscope (AFM) as a model for investigating nonspecific intermolecular interactions between macromolecules in a semidilute region at the single molecule level. Small amount of poly(ethylene glycol) (PEG) terminated with a thiol group was inserted in poly(acrylamide) gels, and a part of PEG polymer segments on the gel surface was attempted to pull out of the gels with a gold-coated AFM tip. The observed force-distance curves were classified into two kinds of extraction force profiles: a plateau force, which is almost constant irrespective of the tip-surface distance and a nonlinear force, which nonlinearly increases with the extraction length. Characteristic interaction length, L, and force, F, of these extraction force profiles were measured with changing the crosslinker concentration of gels which strongly affects the network structures. As a result, L of these extraction profiles significantly decreased at crosslinker concentrations higher than a standard one at which most gels have been prepared for investigating their physical properties. On the other hand, F showed no obvious difference on the change in crosslinker concentrations both on the plateau and the nonlinear force profiles. The origin of the observed forces was discussed in terms of gel network structures.

DNA microstructure based on self-assembly of 4-sticky-end holiday junctions in aqueous solution
Hiroaki Kii, Takuya Takagi, Akira Sasaki, Takaharu Okajima, Masataka Kinjo
JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY, 7, 3, 726, 729, AMER SCIENTIFIC PUBLISHERS, 2007年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Liverwort-like DNA microscale structures consist of 4-sticky-end Holiday junctions as DNA bricks that can be used in nanotechnology and nanobiotechnology to direct the self-assembly of nanomachines as well as DNA assembly. Previously it has not been possible to obtain such DNA microscale structural forms, but herein we report construction of a mesh-like material made up of 4 strands of 40-base DNA. Advanced bioimaging techniques such as fluorescence correlation spectroscopy (FCS), laser scanning microscopy (LSM), and atomic force microscopy (AFM) help us as ultrasensitive detection tools for examing structures in solutions. Combinations of these techniques allow us to survey various chemical conditions of materials and solutions.

Stress relaxation of HepG2 cells measured by atomic force microscopy
T. Okajima, M. Tanaka, S. Tsukiyama, T. Kadowaki, S. Yamamoto, M. Shimomura, H. Tokumoto
NANOTECHNOLOGY, 18, 8, 084010, IOP PUBLISHING LTD, 2007年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Stress relaxation of HepG2 cells was examined with an atomic force microscope ( AFM). In the measurement, a loading force was applied to the cell by an AFM tip, and a time series of the cantilever deflection signal was measured at a fixed position of the cantilever base displacement. The relaxation of the loading force was clearly observed on the HepG2 cells, and was well fitted to a stretched exponential function known as the Kohlrausch-Williams-Watts ( KWW) function, which is empirically employed to represent dispersion processes of the system. The relaxation time and the stretching exponent parameter were estimated to be similar to 0.5 s and 0.4 - 0.6, respectively. The latter indicated that the relaxation observed in HepG2 cells consisted of multiple relaxation processes. Moreover, it was found that the characteristic feature of the relaxation process was not strongly correlated with the elastic properties of the cells.

Nano-dynamics of soft-matter surfaces investigated with an AFM noise analysis　　　　　　　　　　　　　　　
Takaharu Okajima, Tsubasa Kadowaki, Xiang-Ming Tao, Hiroshi Tokumoto
Polymer Preprints, Japan, 55, 2, 3091, 3092, 2006年
日本語, 研究論文（国際会議プロシーディングス）, Knowledge of surface dynamics of softmaterials is crucially important for understanding adhesion, friction and lubrication of polymer and biological systems such as polymer brush, polymer gels and living cells. In the present study, we applied the single molecule spectroscopy and an AFM noise analysis to investigate nano-dynamics of polymer gels in liquids. In the single molecule experiments, a small amount of polymer chains inserted in gels was used as a probe to investigate the gel network structure in terms of nanomechanics. In the noise analysis, on the other hand, the thermal fluctuation of an AFM cantilever is monitored in noncontact and slightly contact to the sample surface as a function of the tip-surface distances, and the power spectrum of cantilevers are analyzed using a harmonic oscillator model with a memory function of the friction coefficient. We will discuss the applicability of these two measurement techniques for investigating dynamics of soft-material surfaces.

Nano-dynamics of living cell surface by AFM　　　　　　　　　　　　　　　
Takaharu Okajima, Masaru Tanaka, Shusaku Tsukiyama, Sadaaki Yamamoto, Masatsugu Shimomura, Hiroshi Tokumoto
Polymer Preprints, Japan, 55, 1, 2109, 2006年
日本語, 研究論文（国際会議プロシーディングス）, Dynamics of living cell surface, hepatocyte was measured with an atomic force microscope (AFM). A thermal fluctuation spectroscopy, in which the thermal noise of cantilever is monitored to estimate the mechanical properties of sample surfaces, was for the first time applied to living cells. The power spectrum of cantilevers was analyzed using a harmonic oscillator with memory function of the friction coefficient. The results showed that the friction coefficient decreased with increasing the frequency in the range of 1-20 kHz, indicating the dispersion of the cell surface friction coefficient in the microsecond time domain.

Quantitative measurements of spatial correlation of polymer gel surfaces with AFM　　　　　　　　　　　　　　　
Tsubasa Kadowaki, Takaharu Okajima, Hiroshi Tokumoto
Polymer Preprints, Japan, 55, 1, 1030, 2006年
日本語, 研究論文（国際会議プロシーディングス）, Surface topography and elasticity of polyacrylamide gels with different crosslinker concentrations were investigated using an atomic force microscope (AFM). In order to measure quantitatively the surface properties, microbeads with 5-60 um in diameter were employed as a probe for force volume experiments instead of a sharp AFM tip. The results showed that with increasing the crosslinker concentration the Young's modulus increases and its spatial distribution broadens. The spatial correlations between the mechanics and the surface structures will be presented.

Nano-measuring living cell surface by AFM　　　　　　　　　　　　　　　
Takaharu Okajima, Masaru Tanaka, Katsuhiro Ishii, Hiroshi Tokumoto
Polymer Preprints, Japan, 54, 1, 2376, 2005年
日本語, 研究論文（国際会議プロシーディングス）, An atomic force microscope (AFM) is a powerful tool for investigating soft materials in liquid environments. We developed a newly designed AFM, which employs a fiber-optic interferometer and a low coherence laser such as a super-luminescence diode. The advantage of this AFM is that this AFM is set up on a conventional optical microscope without blocking a light path for obtaining optical images and that the AFM does not suffer from a stray light of the fiber-optics, which is usually inevitable. It was demonstrated that the AFM could measure the small deflection of the AFM cantilever. Moreover, we developed a technique that precisely measures the contact point between the soft cell surfaces and an AFM tip. The result was that the spectrum of the thermally vibrated cantilever was drastically decreased when the tip contacted the surface, indicating that the method is useful for determining the contact of the tip-soft materials such as living cells in liquids.

Dynamics of a partially stretched protein molecule studied using an atomic force microscope　　　　　　　　　　　　　　　
Takaharu Okajima, Hideo Arakawa, Mohammad, Taufiq Alam, Hiroshi Sekiguchi, Atsushi Ikai
Biophysical Chemistry, 107, 51, 61, 2004年, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Frequency shift feedback imaging in liquid for biological molecules　　　　　　　　　　　　　　　
Hiroshi Sekiguchi, Takaharu Okajima, Hideo Arakawa, Sumihiro Maeda, Akihiko Takashima, Atsushi Ikai
Applied Surface Science, 210, 61, 67, 2003年, ［査読有り］
英語

Nano-mechanical methods in biochemistry using atomic force microscopy　　　　　　　　　　　　　　　
Ikai, A, Afrin, R, Sekiguchi, H, Okajima, T, Alam, M.T, Nishida, S
Current Protein and Peptide Science, 4, 2003年, ［査読有り］
英語

Self-oscillation technique for AFM in liquids　　　　　　　　　　　　　　　
Takaharu Okajima, Hiroshi Sekiguchi, Hideo Arakawa, Atsushi Ikai
Applied Surface Science, 210, 68, 72, 2003年, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Non-destructive force measurement in liquid using atomic force microscope　　　　　　　　　　　　　　　
Hiroshi Sekiguchi, Hideo Arakawa, Takaharu Okajima, Atsushi Ikai
Applied Surface Science, 188, 489, 2002年
英語

Volume Phase Transition in Neutral Copolymer Gels　　　　　　　　　　　　　　　
Takaharu Okajima, Shunsuke Hirotsu
Transactions of the Materials Research Society(!{ Japan, 2001年, ［査読有り］
英語

Use of AFM for imaging and measurement of the mechanical properties of light-convertible organelles in plants　　　　　　　　　　　　　　　
Takafumi Yamada, Hideo Arakawa, Takaharu Okajima, Takayoshi Shimada, Atsushi Ikai
Ultramicroscopy, 91, 261, 268, 2001年, ［査読有り］
英語

Discontinuous Crossover between Fast and Slow Kinetics at the Volume Phase Transition in Poly-N-isopropylacrylamide Gels.
Okajima Takaharu, Harada Ichiro, Nishio Kazufumi, Hirotsu Shunsuke
Japanese Journal of Applied Physics, 39, 8, L875, L877, 公益社団法人 応用物理学会, 2000年
英語, The kinetics of the volume phase transition in N-isopropylacrylamide gels has been studied as a function of crosslinker concentration. The shrinking kinetics at the transition is extremely slow in gels with a standard composition which have been used widely in various experiments. A discontinuous crossover from slow to much faster kinetics were observed in both high- and low-crosslinker-concentration regions, where the characteristic time of the shrinking process changes abruptly by two to four orders of magnitude with a minute change of the crosslinker concentration. In spite of these marked changes in the kinetics, no anomaly was observed in the degree of equilibrium swelling in these regions. A mechanism leading to sudden changes in kinetics is discussed in terms of an inhomogeneous network structure which is dependent on crosslink density.

Study of shear force between glass microprobe and mica surface under controlled humidity　　　　　　　　　　　　　　　
T Okajima, S Hirotsu
Applied Physics Letters, 71, 545, 547, 1997年, ［査読有り］, ［筆頭著者, 責任著者］
英語

Brillouin scattering study of the volume phase transition in poly-N-isopropylacrylamide gels
Shunsuke Hirotsu, Ikuo Yamamoto, Atsushi Matsuo, Takaharu Okajima, Hidemitsu Furukawa, Tatsuyuki Yamamoto
Journal of the Physical Society of Japan, 64, 8, 2898, 2907, Physical Society of Japan, 1995年
英語, 研究論文（学術雑誌）, Brillouin scattering spectra of poly-N-isopropylacrylamide (NIPA) gels swollen to equilibrium in water were measured as a function of temperature both in the swollen and the shrunken phases. This is the first Brillouin scattering experiment on a gel undergoing a volume phase transition. At the discontinuous volume phase transition occuring at 33.6°C, large changes were observed in both of the Brillouin shift and the spectral width. From the measured spectra and the temperature-dependent refractive index of the gel, the hypersonic velocity and the attenuation were determined as a function of temperature. The results are analyzed in terms of the nature of sound propagation in gels, structure of the shrunken phase, relaxation processes of the network and solvent, and a role played by water molecules at the phase transition.

Change in molecular orientation during thin film growth by evaporation of copolymer of vinylidene fluoride (80%) and tetrafluoroethylene (20%)　　　　　　　　　　　　　　　
Takaharu Okajima, Kunisuke Maki
Japanese Journal of Applied Physics, 30, L2055, 1991年, ［査読有り］
英語

Nanoscale Cell Membrane Fluctuations Measured by Scanning Ion Conductance Microscopy
Yusuke Mizutani, Zen Ishikura, Myung-Hoon Choi, Sang-Joon Cho, Takaharu Okajima, BIOPHYSICAL JOURNAL, 104, 2, 317A, 317A, 2013年01月, ［査読有り］
CELL PRESS, 英語, 研究発表ペーパー・要旨（国際会議）

OS1103 原子間力顕微鏡による過渡状態の多数細胞力学測定
水谷 祐輔, 蔡 萍根, 土屋 雅博, 岡嶋 孝治, M&M材料力学カンファレンス, 2012, "OS1103, 1"-"OS1103-2", 2012年09月22日
Mechanical measurements of single cells are crucial for understanding various cell behaviors. The viscoelastic properties of a number of rat fibroblast cells in the process of transformations such as growth (Phase II), senescence (Phase III) and immortalization (IM) were investigated by atomic force microscopy (AFM) combining with a cell microarray technique. The complex shear modulus of cells as a function of frequency was fitted to a power-law model, consisting of a single power-law function in addition to the Newtonian viscosity. Both the mean value and standard deviation of the power-law exponent a strongly depended on the cell phases. As a result, the power-law behaviors of cells were changed in the process of transformation. In addition, the behaviors were strongly dependent on F-actin microfilaments., 一般社団法人日本機械学会, 日本語

1101 原子間力顕微鏡による心筋細胞の拍動挙動の計測(OS13:筋細胞のエンジニアリング)
水谷 祐輔, 土屋 雅博, 河原 剛一, 岡嶋 孝治, バイオエンジニアリング講演会講演論文集, 2009, 22, 187, 187, 2010年01月08日
一般社団法人日本機械学会, 日本語

25pPSB-58 蛍光相関分光法による生細胞膜分子の拡散過程の測定(ポスターセッション,領域12,ソフトマター物理,化学物理,生物物理)
我妻 邦浩, 新倉 謙一, 神谷 亮介, 春菜 ゆかり, 水谷 祐輔, 徳本 洋志, 河原 剛一, 岡嶋 孝治, 日本物理学会講演概要集, 63, 1, 384, 384, 2008年02月29日
一般社団法人日本物理学会, 日本語

20pPSA-44 高強度ソフト&ウェットマター(2) : DNゲルのNecking現象(領域12ポスターセッション,領域12,ソフトマター物理,化学物理,生物物理)
川内 保範, 田中 良巳, 古川 英光, 岡嶋 孝治, 羅 亮皓, 黒川 孝幸, 襲 剣萍, 長田 義仁, 日本物理学会講演概要集, 62, 1, 366, 366, 2007年02月28日
一般社団法人日本物理学会, 日本語

18aTC-8 原子間力顕微鏡による生細胞の力学緩和測定(生物物理,領域12,ソフトマター物理,化学物理,生物物理)
岡嶋 孝治, 田中 賢, 築山 周作, 門脇 翼, 山本 貞明, 下村 政嗣, 徳本 洋志, 日本物理学会講演概要集, 62, 1, 325, 325, 2007年02月28日
一般社団法人日本物理学会, 日本語

超高強度ゲルの創製[XV]DNゲルのNecking現象と破壊メカニズム
川内保範, 田中良巳, 古川英光, 岡嶋孝治, NA Yang-ho, 黒川孝幸, GONG Jian Ping, GONG Jian Ping, 長田義仁, 高分子学会予稿集(CD-ROM), 56, 1 Disk1, 2007年

アクチン集合体形成の物理的メカニズム
KWON Hyuckjoon, 敷中一洋, 角五彰, 田中良巳, 岡嶋孝治, 古川英光, GONG Jian Ping, 長田義仁, コロイドおよび界面化学討論会講演要旨集, 59th, 2006年

アクチン集合体形成の物理的原理
KWON Hyuckjoon, 敷中一洋, 角五彰, 田中良巳, 岡嶋孝治, 古川英光, GONG Jian Ping, GONG Jian Ping, 長田義仁, 高分子学会予稿集(CD-ROM), 55, 2 Disk1, 2006年

原子間力顕微鏡による発生胚皮質領域の力学測定　　　　　　　　　　　　　　　
岡嶋孝治
膜、47、269-272(2022)., 2022年08月

原子間力顕微鏡：多細胞系の力学物性計測の現状　　　　　　　　　　　　　　　
岡嶋孝治
生物物理（解説） 62、159-164(2022), 2022年01月

ナノ計測 : 電子線・光・プローブ技術を用いたナノ・バイオ材料の探索と評価
重川, 秀実, 吉村, 雅満, 目良, 裕, 岡嶋, 孝治
近代科学社, 2020年08月, 9784764950290, x, 179p, 図版 [4]p, 日本語

原子間力顕微鏡法：細胞表面近傍のナノメカニクス　　　　　　　　　　　　　　　
藤井裕紀, 岡嶋孝治
表面と真空、63、437-440 (2020), 2020年

AFMによる多細胞系の力学測定　　　　　　　　　　　　　　　
藤井裕紀, 岡嶋孝治
高分子、68、656-657, 2019年12月, ［分担執筆］

AFMを用いた1細胞・細胞集団の力学物性計測　　　　　　　　　　　　　　　
藤井裕紀, 岡嶋孝治
生体の科学（特集：メカノバイオロジー）vol.70, No.4, 2019年07月, ［分担執筆］

細胞ナノレオロジー：原子間力顕微鏡法　　　　　　　　　　　　　　　
岡嶋 孝治
月刊ソフトマター, 2018年09月, ［分担執筆］

走査型プローブ顕微鏡　　　　　　　　　　　　　　　
淺川雅, 岡嶋孝治, 大西洋
日本分析化学会編、分析化学実技シリーズ機器分析編15, 2017年, ［共著］

原子間力顕微鏡による1細胞レオロジーの定量計測─細胞の個性とエルゴード性　　　　　　　　　　　　　　　
岡嶋 孝治
応用物理学会誌、86、1052-1056, 2017年, ［単著］

Nanorheology of Living Cells　　　　　　　　　　　　　　　
T. Okajima, The World of Nano-Biomechanics (Chapter 13, A. Ikai)
Elsevier, 2016年, ［分担執筆］

原子間力顕微鏡を用いた細胞の力学特性計測　　　　　　　　　　　　　　　
岡嶋孝治, 高橋亮輔, 細胞の特性計測・操作と応用（組織工学ライブラリ－マイクロロボティクスとバイオの融合－ 1）（第1章・第3節）
コロナ社, 2016年, ［分担執筆］

Atomic Force Microscopy: Imaging and Rheology of Living Cells　　　　　　　　　　　　　　　
Takaharu Okajima
Nano/Micro Science and Technology in Biorheology: Principles, Methods, and Applications Chapter 15 (387-414), Springer, (2015), 2015年, ［共著］

原子間力顕微鏡を用いた細胞レオロジー特性の計測　　　　　　　　　　　　　　　
岡嶋 孝治
三次元ティッシュエンジニアリング～細胞の培養・操作・組織化から品質管理、脱細胞化まで～（第1章・第3節）、（株式会社NTS） (2015), 2015年, ［共著］

イオンコンダクタンス顕微鏡：細胞膜表面の揺らぎを計測する　　　　　　　　　　　　　　　
水谷祐輔, 田中良昌, 田中あや, 住友弘二, 岡嶋孝治
光学-細胞機能に迫る非染色非破壊イメージング技術、44, (6月号）(2015), 2015年, ［共著］

High-throughput measurements of single cell rheology by atomic force microscopy　　　　　　　　　　　　　　　
K. Kuribayashi-Shigetomi, R. Takahashi, A. Subagyo, K. Sueoka, T. Okajima
Hyper Bio Assembler for 3D Cellular Systems, Chapter 4 (57-67), Springer, 2015, 2015年, ［共著］

原子間力顕微鏡による超高速細胞メカニクス計測技術　　　　　　　　　　　　　　　
高橋亮輔, 岡嶋孝治
ケミカルエンジニヤリング（先端計測技術開発の展望:9月号）(2015), 2015年

細胞の物理学：ＳＰＭによる定量計測　　　　　　　　　　　　　　　
岡嶋 孝治
表面科学 35, 544-544(2014), 2014年, ［共著］

走査プローブ顕微鏡による細胞物性測定：原子間力顕微鏡とイオンコンダクタンス顕微鏡（総説）　　　　　　　　　　　　　　　
岡嶋孝治
化学工業 (2013), 2013年

原子間力顕微鏡による細胞力学の定量計測（解説）　　　　　　　　　　　　　　　
岡嶋 孝治
検査技術 (2013), 2013年

原子間力顕微鏡による細胞レオロジー測定（総説）　　　　　　　　　　　　　　　
岡嶋孝治
日本膜学会「膜」38, 76-81 (2013), 2013年

1細胞レオロジー測定 : 原子間力顕微鏡による細胞レオロジー計測の新手法　　　　　　　　　　　　　　　
岡嶋 孝治, 水谷 祐輔
生物物理 52, 5, 230-233, 2012年09月

細胞力学計測のためのマイクロ加工基板を用いた原子間力顕微鏡法　　　　　　　　　　　　　　　
岡嶋 孝治, 水谷 祐輔
表面科学33(8) 461-466, 2012年08月

１細胞レオロジー測定：原子間力顕微鏡による細胞レオロジー計測の新手法（総説）　　　　　　　　　　　　　　　
岡嶋孝治, 水谷祐輔
日本生物物理学会誌「生物物理」52, 230-233 (2012), 2012年

走査プローブ顕微鏡（実験物理科学シリーズ6）　　　　　　　　　　　　　　　
共立出版, 2009年

原子間力顕微鏡による生細胞ナノレオロジー計測　　　　　　　　　　　　　　　
岡嶋孝治, 徳本洋志
日本レオロジー学会誌 vol.36, No.2, p. 81-86, 2008年04月, ［分担執筆］

ナノテクのための物理入門　　　　　　　　　　　　　　　
共立出版, 2007年

BioNics[バイオニクス]　　　　　　　　　　　　　　　
オーム社, 2005年

ソフトナノテクノロジー —バイオマテリアル革命—　　　　　　　　　　　　　　　
シーエムシー出版, 2005年

Mapping mechanical properties of single cells during embryogenesis using atomic force microscopy　　　　　　　　　　　　　　　
T. Okajima
AFM BioMED conference, (2022.8.29-9.2, Okazaki）, 2022年12月, 英語, 口頭発表（招待・特別）
［招待講演］

多細胞メカニクスの時空間マッピング測定　　　　　　　　　　　　　　　
岡嶋 孝治
ナノプローブテクノロジー第167委員会 第103回研究会、高分子×SPM（2022.7.27、大岡山）, 2022年07月, 日本語, 口頭発表（招待・特別）
［招待講演］

原子間力顕微鏡による発生胚の力学動態の計測　　　　　　　　　　　　　　　
岡嶋 孝治
第61回日本生体医工学会、細胞アッセイ：細胞動態の把握と計測、（2022.6.28-30、新潟）, 2022年06月, 日本語, 口頭発表（招待・特別）
［招待講演］

発生胚表面のメカニクス：原子間力顕微鏡測定　　　　　　　　　　　　　　　
岡嶋孝治
日本膜学会の第44回年会、（2022.6.9-10、早稲田）, 2022年06月, 日本語, 口頭発表（招待・特別）
［招待講演］

Nanomechanics of single cells and tissues revealed by atomic force microscopy　　　　　　　　　　　　　　　
T. Okajima
Pacifichem 2021, Experimental and Computational Analysis of the Nano-Bio Interface for Sustainable Nanotechnology, (Dec.16-21, 2021, Online), 2021年12月, 英語, 口頭発表（招待・特別）

Measuring mechanical properties of developing embryo with AFM　　　　　　　　　　　　　　　
T. Okajima
MRS Fall meeting 2021, Frontiers in Scanning Probe Microscopy—Beyond Imaging of Soft Materials, （2021.12.6-8, Boston/Online Hybrid), 2021年12月, 英語, 口頭発表（招待・特別）
［招待講演］

発生胚のAFM計測：現状と課題　　　　　　　　　　　　　　　
岡嶋孝治
第17回バイオオプティクス研究会（2021年12月10-11日、山口/オンライン）, 2021年12月, 日本語, 口頭発表（招待・特別）
［招待講演］

AFMによる受精卵の発生過程の力学計測　　　　　　　　　　　　　　　
岡嶋孝治
日本顕微鏡学会 第77回学術講演会、先端ナノプローブ法による高分解能局所物性・オペランド計測（2021年6月14日、つくば）, 2021年06月, 日本語, 口頭発表（招待・特別）
［招待講演］

AFMによる発生胚のメカニクス　　　　　　　　　　　　　　　
岡嶋孝治
2021年応物春季学術講演会, 革新的走査型プローブ顕微鏡技術で拓くナノプローブ生命科学の新展開（2021.3.16-19、onine）, 2021年03月, 日本語, 口頭発表（招待・特別）
［招待講演］

原子間力顕微鏡：細胞・組織のメカニクス測定　　　　　　　　　　　　　　　
岡嶋孝治
日本薬剤学会・物性FGセミナー2020・顕微鏡法で医薬品原薬・製剤を“顕わ”にする―低分子から細胞まで―、2021.2.19、オンライン, 2021年02月, 日本語, 口頭発表（招待・特別）
［招待講演］

Nanomechanics of cell and tissue probed by atomic force microscopy　　　　　　　　　　　　　　　
T. Okajima
6th International Conference on Electronic Materials and Nanotechnology for Green Environment (ENGE 2020) (2020. 11.1-4, Korea_online), 2020年11月, 英語, 口頭発表（招待・特別）
［招待講演］

AFMによる細胞・組織の力学情報の定量化　　　　　　　　　　　　　　　
岡嶋孝治
2020年第81回応用物理学会秋季学術講演会・多次元計測技術とデータサイエンスの融合によるバイオイメージング・センシング技術の進展（京都オンライン、2020.9.9）, 2020年09月, 日本語, 口頭発表（招待・特別）
［招待講演］

バイオAFM計測：1細胞から多細胞へ　　　　　　　　　　　　　　　
岡嶋孝治
第59回日本生体医工学会大会（岡山オンライン、2020.5.25-27）, 2020年05月, 日本語, 口頭発表（招待・特別）
［招待講演］

原子間力顕微鏡による細胞・組織メカニクスの定量化　　　　　　　　　　　　　　　
岡嶋孝治
第67回応用物理学会春期学術講演会・多次元計測技術とデータサイエンスの融合によるバイオイメージング・センシングの将来、2020年3月12日-15日、東京, 2020年03月, 日本語, 口頭発表（招待・特別）
［招待講演］

AFMによる1細胞・多細胞系のナノ力学計測　　　　　　　　　　　　　　　
岡嶋孝治
2019年日本表面真空学会学術講演会・局所分光とバイオ計測（プローブ顕微鏡部会）（つくば、2019.10.28-30）, 2019年10月, 日本語, 口頭発表（招待・特別）
［招待講演］

AFMによる多細胞系のメカニクス　　　　　　　　　　　　　　　
岡嶋孝治
日本顕微鏡学会 ソフトマテリアル研究部会 第6回講演会（宮城蔵王、2019.10.25-26）, 2019年10月
［招待講演］

AFMよる1細胞・細胞集団のナノメカニクス　　　　　　　　　　　　　　　
岡嶋孝治
日本顕微鏡学会第75回学術講演会・原子間力顕微鏡技術のパラダイムシフト～原子から細胞まで階層をまたぐ構造・機能解析～（名古屋、2019.6.17-19）, 2019年06月, 日本語, 口頭発表（招待・特別）
［招待講演］

階層性自己組織化複合材料デザイン（代表者：松本卓也）　　　　　　　　　　　　　　　
CREST
2022年10月 - 2028年03月
岡嶋孝治
JST, 分解・劣化・安定化の精密材料科学, 研究分担者, JPMJCR22L5

プローブ顕微鏡による発生胚の遺伝子・力学的相互作用の解明
科学研究費助成事業
2024年04月01日 - 2027年03月31日
岡嶋 孝治, 道上 達男, 繁富 香織
日本学術振興会, 基盤研究(A), 北海道大学, 24H00412

原子間力顕微鏡による1細胞診断技術の高速化　　　　　　　　　　　　　　　
基盤研究(B)
2021年04月 - 2024年03月
岡嶋 孝治
研究代表者

組織弾性モダリティの構築と老化制御
科学研究費助成事業
2021年07月09日 - 2023年03月31日
早野 元詞, 岡嶋 孝治
日本学術振興会, 挑戦的研究(萌芽), 慶應義塾大学, 21K18608

原子間力顕微鏡を用いた発生胚メカニクスの包括的マッピング計測技術の開発　　　　　　　　　　　　　　　
挑戦的研究(萌芽)
2021年04月 - 2023年03月
岡嶋 孝治
研究代表者

原子間力顕微鏡を用いた肺高血圧症のin vitro薬剤反応評価システムの構築
科学研究費助成事業
2019年04月01日 - 2022年03月31日
小垣 滋豊, 岡嶋 孝治, 石田 秀和
特発性肺動脈性肺高血圧症患者の肺移植時に採取した肺動脈平滑筋細胞とドナー肺のトリミングで出た余剰組織から樹立した肺動脈平滑筋細胞とを用いて、原子間力顕微鏡により、細胞の機械的特性について検討した。これまで臨床応用されている薬物を用いて、薬剤を平滑筋細胞に添加した場合に、細胞のレオロジーがどのように変化するか検討した。代表的なPDE5阻害薬であるシルデナフィルでは、肺高血圧症患者の肺動脈平滑筋細胞のみ細胞弾性率が低下した。マシテンタンおよびリオシグアトを添加すると、用量依存的に細胞弾性率の低下と細胞流動性の上昇がみられた。両者を同時に添加すると、より低い薬物濃度で弾性率の低下が認められた。
日本学術振興会, 基盤研究(C), 大阪大学, 19K08276

マルチプローブ顕微鏡技術を用いた発生胚メカニクス計測法の開発　　　　　　　　　　　　　　　
挑戦的研究（萌芽）
2019年07月 - 2021年03月
岡嶋 孝治
研究代表者, 競争的資金

細胞のエルゴード性を利用したがん細胞力学診断・アッセイ法の開発　　　　　　　　　　　　　　　
基盤研究(B)
2018年04月 - 2021年03月
岡嶋 孝治
研究代表者, 競争的資金

原子間力顕微鏡を用いた中枢肺動脈のレオロジー解析による肺高血圧症の病態解明
科学研究費助成事業
2016年04月01日 - 2019年03月31日
成田 淳, 岡嶋 孝治, 桂木 慎一
肺動脈性肺高血圧症においては、末梢肺動脈における抵抗（レジスタンス）だけではなく、中枢肺動脈におけるコンプライアンスも重要であることが近年理解されてきた。しかしこれまで肺動脈平滑筋細胞における粘弾性が、肺高血圧症においてどのような役割を果たしているのかは確立されていない。本研究では、肺高血圧症患者から得た肺動脈平滑筋細胞において原子間力顕微鏡を用いることでその粘弾性を精度高く計測することが可能となった。肺高血圧症患者においては、粘弾性の高い細胞の一群が存在し、肺血管拡張薬投与によって、その粘弾性は低下することが明らかとなった。
日本学術振興会, 基盤研究(C), 大阪大学, 16K10065

原子間力顕微鏡を用いた発生胚メカニクスの網羅解析法の開発　　　　　　　　　　　　　　　
挑戦的研究(萌芽)
2017年04月 - 2019年03月
岡嶋 孝治
研究代表者, 競争的資金

ヒト間葉系幹細胞治療技術の開発とその最適化に関する研究　　　　　　　　　　　　　　　
二国間交流事業 日本—シンガポール 共同研究
2015年04月 - 2017年03月
岡嶋 孝治
研究代表者, 競争的資金

多重周波数フォースモジュレーション顕微鏡の開発：細胞レオロジー変数のイメージング　　　　　　　　　　　　　　　
挑戦的萌芽研究
2015年04月 - 2017年03月
岡嶋 孝治
研究代表者, 競争的資金

原子間力顕微鏡による生体組織力学物性のその場分離計測技術の開発　　　　　　　　　　　　　　　
新学術領域研究(研究領域提案型)バイオアセンブラ
2014年04月 - 2016年03月
岡嶋 孝治
研究代表者, 競争的資金

プローブ顕微鏡による細胞間力学的相互作用の時空間揺らぎの研究　　　　　　　　　　　　　　　
新学術領域研究(研究領域提案型) ゆらぎと構造
2014年04月 - 2016年03月
岡嶋 孝治
研究代表者, 競争的資金

イオンコンダクタンス顕微鏡を用いた細胞膜揺らぎの定量イメージング技術の開発　　　　　　　　　　　　　　　
挑戦的萌芽研究
2013年04月 - 2015年03月
岡嶋 孝治
研究代表者, 競争的資金

原子間力顕微鏡を用いた細胞力学伝播関数の時空間定量解析　　　　　　　　　　　　　　　
基盤研究(B)
2013年 - 2015年
岡嶋 孝治
研究代表者, 競争的資金

原子間力顕微鏡による超高速細胞レオロジー分離技術の開発　　　　　　　　　　　　　　　
新学術領域研究(研究領域提案型)バイオアセンブラ
2012年04月 - 2014年03月
岡嶋孝治
研究代表者, 競争的資金

原子間力顕微鏡を用いたがん細胞力学診断技術の開発　　　　　　　　　　　　　　　
2011年04月 - 2013年03月
岡嶋 孝治
挑戦的萌芽研究, 研究代表者, 競争的資金

原子間力顕微鏡法を用いたがん細胞診断自動解析法の開発　　　　　　　　　　　　　　　
研究成果最適展開支援プログラム（A-STEP）
2012年 - 2013年
岡嶋 孝治
研究代表者, 競争的資金

原子間力顕微鏡を用いた多数細胞力学の多変量解析の研究　　　　　　　　　　　　　　　
2009年04月 - 2012年03月
岡嶋 孝治
基盤研究(B), 研究代表者, 競争的資金

細胞周期制御下の細胞界面ナノダイナミクスの研究　　　　　　　　　　　　　　　
2009年04月 - 2011年03月
岡嶋 孝治
特定領域研究・高次系分子科学, 研究代表者, 競争的資金

走査プローブ顕微鏡と蛍光相関分光法による生細胞界面の1分子ダイナミクスの研究　　　　　　　　　　　　　　　
2008年04月 - 2010年03月
岡嶋 孝治
特定領域研究・高次系分子科学, 研究代表者, 競争的資金

単一細胞表層の全方向ナノダイナミクス計測技術の開発　　　　　　　　　　　　　　　
産業技術研究助成事業
2006年 - 2009年
岡嶋孝治
NEDO, 産業技術研究助成事業, 研究代表者

糖鎖1分子のナノ力学刺激に対する応答ダイナミクスの研究　　　　　　　　　　　　　　　
2005年04月 - 2007年03月
岡嶋 孝治
萌芽研究, 研究代表者, 競争的資金

細胞内部探索用カーボンナノチューブAFM技術の研究
科学研究費助成事業
2004年 - 2006年
徳本 洋志, 岡嶋 孝治, 畔原 宏明
表面科学分野の研究において多大の貢献をしてきた原子問力顕微鏡(AFM)の今後の大きな応用分野は、溶液中での動作することを生かし溶媒中にある単一の細胞や蛋白質の形状を分子レベルの分解能で直接観察しバイオナノテクノロジー分野への展開を図ることである。更に、細胞内部の構造や機能を計測することができるようになると、バイオ材料分野の研究から生命科学の分野への展開も図ることが出来る。
本研究では、将来、細胞内部を探索できるカーボンナノチューブAFM技術の要素技術として、先端形状がナノスケールで機械的・化学的に最も安定した材料であるカーボンナノチューブ(CNT)の一本を市販のシリコンAFM探針先端に接着する技術、モデル細胞膜としてDMPC分子を用いた人工脂質二分子膜の作製技術、さらに前者を後者に挿入する技術を確立した。また、CNT探針先端にカルボン酸で化学修飾し、試料としてマイクロコンタクトプリンティング法により作製した親・疎水性パターンを両者間に働く微弱な分子間力のAFM計測を通して明確にした。これにより、化学種を識別する技術(化学力AFM技術)の基礎を確立し、DNA塩基読み取り技術への見通しを得た。
一方、細胞内外には多種多様のバイオ高分子が存在しており、AFM探針との間に非特異的な相互作用をする。この非特異的な相互作用を計測するため、高分子ゲルの表面あるいは内部にポリエチレングリコール1分子を接着・埋め込み両者間の相互作用力の測定を行いそれぞれに固有の非線形力を得た。更に、1分子の方向を規定してマニピュレーションする技術の基礎の確立にも成功した。また、ヒト肝細胞に探針を押し付けた後の応力緩和測定から細胞の動的な粘弾性に関する知見が得られること見出した。これらの成果により、細胞の構造と機能を理解するための力学的手法による基礎が確立できた。
日本学術振興会, 基盤研究(B), 北海道大学, 16310078

生体分子のマイクロ秒粘弾性測定　　　　　　　　　　　　　　　
2003年04月 - 2005年03月
岡嶋 孝治
若手研究(B), 研究代表者, 競争的資金

原子間力顕微鏡を用いた単一高分子鎖の相転移における力学緩和の測定　　　　　　　　　　　　　　　
若手研究(B)
1999年04月 - 2001年03月
岡嶋 孝治
研究代表者, 競争的資金

シェアフォース顕微鏡におけるプローブ・表面間相互作用の起源の解明　　　　　　　　　　　　　　　
奨励研究(A)
1997年04月 - 1999年03月
岡嶋 孝治
研究代表者, 競争的資金

イオン性ゲルの物性と構造に関する基礎的研究
科学研究費助成事業
1998年 - 1999年
弘津 俊輔, 岡嶋 孝治
体積相転移のメカニズムに関する研究:ゲルの体積相転移に対する従来の現象論を批判的に検討し、2つのゲル相の間の相転移であるという考えが誤りである事を指摘した。従来の考えと異なり、収縮相はゲルではなくて析出網目であるという新しい相転移モデルを提案した。Poly-N-isopropylacrylamideゲルの収縮相の動的光散乱測定および弾性測定を初めて実行し、新たに提案されたモデルを支持する結果を得た。
イオン性ゲルのメソ相形成に関する研究:イオンを含むゲルにおける電荷分離を伴うメソ相形成を光散乱によって初めて検出した。メソ相周期は光の波長よりも遙かに小さいので、これまで光散乱では検出出来ないと考えられていたが、本実験により緩和時間に顕著な以上が見出された。
体積相転移点近傍とスピノーダル点近傍における弾性率および弾性緩和測定:電子天秤と微動電動ステージを用いた弾性率測定装置を開発した。この装置は特に、ゴム状物質やゲルの機械的測定に適し、静的弾性率のほか、応力一定の弾性緩和、ひずみ一定の弾性緩和を共に高精度で測定できる。この装置を用いて、体積相転移を起こして収縮相に入ったゲルの弾性率と弾性緩和を初めて測定し、またスピノーダル分解に伴う弾性以上の存在を初めて見出した。
生体多糖ゲルと合成ゲルとの構造的相違の研究:生体ゲルであるゼラチンおよびκ-carrageenanと合成ゲルであるイオン性アクリルアミド系ゲルとの動的光散乱測定を行い、両者の動的挙動の相違を、解析検討し、両者の架橋構造の相違に対する知見を得た。
日本学術振興会, 基盤研究(B), 東京工業大学, 10440116

温度敏感性ゲルを用いた細胞培養による細胞の応力応答の研究
科学研究費助成事業
1997年 - 1998年
弘津 俊輔, 岡嶋 孝治
本年度に行った研究とその成果を以下にまとめた。
1) 温度敏感性ゲルの相転移を利用した、細胞への応力印加の実験。: これについては、ゼラチンを混入したNIPAゲルを用いて、試行実験を行った。結果は、膨潤相から収縮相への変化に関しては目立った効果は見られなかった。現在、細胞に有効に力を印可する方法を開発すると共に、収縮相から膨潤相への転移の効果も調べつつある。
2) 化学架橋ゼラチンゲル基盤の合成: 細胞接着性が良好で、しかも比較的自由に弾性的性質を調節出来る基盤として、ゼラチンとコハク化ゼラチンの混合物をカルボジイミドで架橋したゲルを開発した。ゼラチンとコハク化ゼラチンの混合比を変えることにより、弾性率が同じで、膨潤率が異なるゲルを得ることが出来た。
3) ゲルの弾性率測定: 電子天秤を利用してゲルに一定の加重を印可し、その変形から、ゲルの静的弾性率(ヤング率、合成率、および体積弾性率)を決定した。
4) ゲル基盤の弾性的性質と培養細胞の形状との関係
基盤(足場)と細胞との力学的相互作用が、細胞の機能、増殖能、形状などに与える影響を理解するためには、基盤の弾性的性質が細胞に与える効果を調べる必要がある。上記の方針で、弾性率を測定したゲルで、マウス3T3繊維芽細胞を培養してその形状を調べた結果、細胞形状は基盤の堅さに強く依存することが分かった。具体的には、基盤ゲルのヤング率が1x10^4[dyn/cm^2]以上の場合、7x10^3〜1x10^4[dyn/cm^2]、7x10^3[dyn/cm^2]以下の場合のそれぞれに応じて、細胞の外径および増殖のパターンに顕著な相違が見られた。より詳しい解析を実行中である。
日本学術振興会, 萌芽的研究, 東京工業大学, 09875243