A comparison of cerebral amyloid angiopathy in the cerebellum and CAA-positive occipital lobe of 60 brains from routine autopsies.

We semiquantitatively compared the frequency and severity of cerebral amyloid angiopathy (CAA) in the cerebellum and CAA-positive occipital lobe of 60 subjects from routine autopsies. In the 60 subjects with a CAA-positive occipital lobe, cerebellar CAA was observed in 29 subjects (48.3%), and the severity of cerebellar CAA was relatively mild compared with occipital lobe CAA. Capillary CAA was observed in the occipital lobe of 12 subjects and the cerebellum of three subjects. CAA-related vasculopathies were observed in the occipital lobe of 15 subjects and the cerebellum of two subjects. The severity of CAA-related vasculopathy was mild in both of these subjects. Amyloid-ß plaques were observed in the occipital lobe of 54 subjects (90%) and the cerebellum of 16 subjects (26.7%). The severity of amyloid-ß plaques in the cerebellum was mild compared with the occipital lobe. In summary, we confirmed that cerebellar CAA is frequently observed in the cerebellum but with a lower severity than CAA in the occipital lobe.

An autopsy case of amyloid angiopathy-related cerebellar hemorrhage.

An 80-year-old man with dementia demonstrated cerebellar hemorrhage. Autopsy revealed pathology compatible with Alzheimer's disease and cerebral amyloid angiopathy (CAA). CAA was more prevalent in the occipital lobe than in the frontal, parietal, and temporal lobes; however, amyloid-ß (Aß)-containing senile plaques were less abundant in the occipital cortex than in the other cortices. In the cerebellum, abundant CAA-involved vessels were observed in the subarachnoid space and molecular layer and to a lesser extent in the Purkinje and granule layers. On consecutive sections, Aß1-42 immunohistochemistry revealed senile plaques and CAA-involved vessels with strong immunoreactivity whereas Aß1-40 immunohistochemistry identfied CAA-involved vessels with strong immunoreactivity and senile plaques with weak immunoreactivity in the cerebellar cortices.

[Seasonal changes in blood pressure and related factors among older patients receiving home medical care].

AIM: We investigated seasonal variations in blood pressure (BP) and factors related to these variations among older patients receiving home medical care. METHOD: A total 57 patients ≥ 65 years old receiving home medical care who participated in the Osaka Home Care REgistry study (OHCARE), a prospective cohort study, were included. We investigated the seasonal patient characteristics and variations in the BP. In addition, to determine the influence of seasonal variations in the systolic blood pressure (SBP) on the occurrence of clinical events (hospitalization, falls and death), we classified patients into larger- and smaller- change groups based on the median seasonal variations in SBP. RESULT: About 60% of subjects were very frail or bedridden. The mean BP was higher in winter than in summer (124.7±11/69.5±7 vs.120.5±12/66.9±8 mmHg) (P< 0.01). On comparing the characteristics of the two groups with larger and smaller changes in the SBP, the group with large BP changes had a significantly lower BP in summer than the group with small BP changes. In addition, the incidence of "hospitalization" was significantly higher in the group with large BP changes than in the group with small BP changes (P = 0.03). CONCLUSION: The present study revealed that there were seasonal changes in the BP in older patients receiving home medical care. It was also suggested that seasonal changes in the BP might be associated with the risk of hospitalization events. Given these BP variations, doctors and visiting nurses should be alert for systemic abnormalities, especially in frail patients receinving home medical care.

Template-based fabrication of spatially organized 3D bioactive constructs using magnetic low-concentration gelation methacrylate (GelMA) microfibers.

Low concentrations of gelatin methacrylate (GelMA) microfibers are more favorable for cellular activity compared with high concentrations. However, applying low-concentration GelMA microfibers as building blocks for higher-order cellular assembly remains challenging owing to their poor mechanical properties. Herein, we report a new template-based method to solve this problem. GelMA microfibers (5%, w/v) containing magnetic nanoparticles were synthesized by a microfluidic spinning method. A 9 × 9 micropillar array surrounded by a magnetic substrate was constructed to form 8 × 8 microgaps arranged in a crisscross pattern as a magnetic template. In DMEM solution, magnetic attraction facilitated efficient arrangement of the microfibers according to the template with micron assembly accuracy, with a microgrid-like construct (microGC) generated after removing all micropillars. MicroGCs were shown to effectively support the activities of surface seeded or encapsulated cells and be flexibly constructed with various organized spatial patterns. Owing to the low mechanical property requirements of assembled microfibers and the easy-to-implement operation, the proposed method provides a versatile pathway for the assembly of various microfluidic spun microfibers. Furthermore, the resulting 3D microgrid-like cellular constructs with organized spatiotemporal composition offer a convenient platform for the study of tissue engineering.

Amyloid-ß plaques may be reduced in advanced stages of cerebral amyloid angiopathy in the elderly.

We examined 29 cases in which cerebral amyloid angiopathy (CAA) was detected among routine aged autopsies. Most cases with severe CAA had many amyloid-ß (Aß) plaques in the occipital cortex. Nonetheless, two cases had few Aß plaques with many small vessels and capillaries with CAA. In the two cases, severe CAA was widely distributed, except in the frontal lobes. Aß deposits in capillaries often showed the characteristic pattern of dysphoric amyloid angiopathy. A few naked plaques were present. Although Aß plaques were sparse near small vessels with CAA, there were many Aß plaques distant from small vessels with CAA. Some of the remaining plaques had a moth-eaten appearance. Based on Aß-positive star-like appearance and results of double immunohistochemistry for glial fibrillary acidic protein and Aß1-42 , some astrocytes appeared to contain Aß. Ionized calcium-binding adapter molecule 1 (Iba1)-positive microglia were scattered within the neuropil, with some present around small vessels with CAA. Iba1-positive microglia also seemed to phagocytose Aß in several senile plaques by double immunostaining. Neurons and neurites identified with a monoclonal antibody against phosphorylated tau (clone AT8) were occasionally detected in sparse plaque areas, with AT8-identified dot-like structures present around capillaries with CAA. Accumulation of T lymphocytes was detected around vessels in the subarachnoid space in one case. The morphological changes detected in our two cases were similar to those of morphological markers of plaque clearance after Aß immunotherapy. Nonetheless, our cases did not receive Aß immunotherapy, but similar pathologies were observed. Overall, advanced CAA cases, including our two cases, may be examples of plaque clearance without Aß immunotherapy. Further studies are needed to resolve the mechanism of Aß plaque clearance using these cases.

Multifunctional Noncontact Micromanipulation Using Whirling Flow Generated by Vibrating a Single Piezo Actuator.

A noncontact method that can achieve immobilization, transportation, and rotation in the microscale is desired in biological micromanipulation. A multifunctional noncontact micromanipulation method is proposed here based on a vibration-generated whirling flow. Resonance of a cantilever structure is utilized to extend the straight vibration of a single piezo actuator to the 2D circular vibration of a micropipette. The circular vibration in fluids can generate the whirling flow featured with low pressure in the core area and flow velocity gradient. The low pressure can immobilize the objects nearby and transport them together with the micropipette, and the flow velocity gradient is utilized to form a torque to rotate the immobilized object. Experiments of the microbeads are conducted to evaluate the claimed functions and quantify the key parameters that influence the rotation velocity. The cell spheroid is immobilized and rotated for 3D observation, and by assessing the viability of the cells containing in the spheroid, the proposed method is proved noninvasive to living cells. Finally, another important application in operations of mouse egg cells is shown, which indicates that the proposed method is a potential valuable tool in biological micromanipulation.

Astrocytic tau pathologies in aged human brain.

Argyrophilic and tau-positive abnormal structures in astrocytes are frequent in aged brains, with a new nomenclature of aging-related tau astrogliopathy (ARTAG) proposed. The two major cytomorphologies of ARTAG are thorn-shaped astrocytes (TSA) and granular or fuzzy tau immunoreactivity in processes of astrocytes (GFA). We selected 28 cases in which many AT8-identified astrocytic tauopathies were observed in the central nervous system from 330 routine aged autopsied cases, including Alzheimer's disease. AT8-identified and Gallyas silver staining-positive TSA were observed in subpial, subependymal, perivascular areas as well as white matter. These TSA were 4-repeat (4R) tau-positive. In contrast, 3-repeat (3R)-tau was negative in TSA, but positive in short thick cell processes, likely neuropil threads, in subpial and subependymal areas. The frequency of 3R-tau-positive processes was variable. Small dot-like AT8-identified astrocytic processes surrounding vessels in the neuropil were also positive for 4R-tau, but negative for 3R-tau. GFA in cerebral gray matter were AT8-identified and Gallyas-positive, and positive for 4R-tau but negative for 3R-tau. In this study, we did not identify 3R-tau+/4R-tau+ or 3R-tau+/4R-tau- astrocytes. Further studies are needed to clarify the nature and progression of glial tau-positive structures in ARTAG.

Multicellular Co-Culture in Three-Dimensional Gelatin Methacryloyl Hydrogels for Liver Tissue Engineering.

Three-dimensional (3D) tissue models replicating liver architectures and functions are increasingly being needed for regenerative medicine. However, traditional studies are focused on establishing 2D environments for hepatocytes culture since it is challenging to recreate biodegradable 3D tissue-like architecture at a micro scale by using hydrogels. In this paper, we utilized a gelatin methacryloyl (GelMA) hydrogel as a matrix to construct 3D lobule-like microtissues for co-culture of hepatocytes and fibroblasts. GelMA hydrogel with high cytocompatibility and high structural fidelity was determined to fabricate hepatocytes encapsulated micromodules with central radial-type hole by photo-crosslinking through a digital micromirror device (DMD)-based microfluidic channel. The cellular micromodules were assembled through non-contact pick-up strategy relying on local fluid-based micromanipulation. Then the assembled micromodules were coated with fibroblast-laden GelMA, subsequently irradiated by ultraviolet for integration of the 3D lobule-like microtissues encapsulating multiple cell types. With long-term co-culture, the 3D lobule-like microtissues encapsulating hepatocytes and fibroblasts maintained over 90% cell viability. The liver function of albumin secretion was enhanced for the co-cultured 3D microtissues compared to the 3D microtissues encapsulating only hepatocytes. Experimental results demonstrated that 3D lobule-like microtissues fabricated by GelMA hydrogels capable of multicellular co-culture with high cell viability and liver function, which have huge potential for liver tissue engineering and regenerative medicine applications.

[Factors associated with behavioral and psychological symptoms in patients with dementia who received home medical treatment: A cross-sectional study (OHCARE study)].

AIM: The present study investigated the behavioral and psychological symptoms of dementia (BPSD) and pharmacological therapy among elderly people with dementia who received home medical treatment. METHODS: This study was part of the Osaka Home Care Registry study (OHCARE-study). Participants were >65 years old with dementia. Demographic and medical data, BPSD, and the LTCI [long-term care insurance] care-need level were collected. A multiple logistic regression analysis was performed in order to clarify the factors associated with BPSD. RESULTS: Among 110 subjects (82.0±11.3 years old), 64.6% had a diagnosis of dementia, most commonly Alzheimer's dementia. Aside from home medical treatment, 58.1% had a nurse visit, 48.1% received home care, and 40.0% used a day service. The prevalence of BPSD was 53.0%. Those with BPSD most frequently had an LTCI care-need level of 3, and the prevalence of BPSD declined as the level increased after 3. Psychotropic drugs were prescribed in 61.5% of those with BPSD. Antipsychotic drugs were used significantly more frequently in those with nursing care resistance, assault and delusions than in others (all, p <0.005). A multivariate analysis showed that the positive predictor of BPSD was antipsychotic drug use, while negative predictors were an LTCI care-need level and the use of visiting rehabilitation. CONCLUSION: The BPSD in elderly dementia patients receiving home medical treatment were clarified. The LTCI care-need level is an independent predictor of BPSD after adjusting for the activities of daily life. Further longitudinal investigations including the BPSD severity and frequency are needed.

Adult moyamoya disease associated with abundant phosphorylated tau accumulation in the brainstem: Report of a case with autopsy findings.

We report the case of a 72-year-old Japanese woman with moyamoya disease (MMD). She experienced her first intracerebral hemorrhage (ICH) at the age of 32 years, and had nine ICHs and/or intraventricular hemorrhages during the following 40 years. Cerebral angiograms and vascular pathologies at autopsy confirmed that the patient suffered from MMD. Macroscopically, there were brown-colored changes in the subarachnoid space, mainly at the base of the brain and around the cerebellar hemispheres. Microscopically, hemosiderin deposits were observed mainly in the old hemorrhagic lesions and on the surface of the brainstem and cerebellum. Many AT8-immunoreactive neurons and neurites were observed in the pons and midbrain, mainly in the locus ceruleus and reticular formation in the midbrain. Several AT8-immunoreactive neurons and neurites were positive for Gallyas silver staining. A few tiny and short AT8-immunoreactive processes were observed in the molecular, Purkinje cell and granular layers of the cerebellum. There were a few phosphorylated tau accumulations in the cerebrum without senile plaques. Lewy pathologies and transactive response DNA-binding protein 43 kDa proteinopathy were not detected. We suspect that oxidative stress after repeated bleedings with long-term courses in the ventricles and subarachnoid space may accelerate phosphorylated tau accumulation in the brainstem. To our knowledge, this is the first report of MMD with tauopathy in the brainstem.

Hydrodynamic Tweezers: Trapping and Transportation in Microscale Using Vortex Induced by Oscillation of a Single Piezoelectric Actuator.

The demand for a harmless noncontact trapping and transportation method in manipulation and measurement of biological micro objects waits to be met. In this paper, a novel micromanipulation method named “Hydrodynamic Tweezers” using the vortex induced by oscillating a single piezoelectric actuator is introduced. The piezoelectric actuator is set between a micropipette and a copper beam. Oscillating the actuator at a certain frequency causes the resonance of the copper beam and extend 1D straight oscillation of the piezoelectric actuator to 2D circular oscillation of the micropipette, which induces a micro vortex after putting the micropipette into fluid. The induced vortex featuring low pressure in its core area can trap the object nearby. A robotic micromanipulator is utilized to transport the trapped objects together with the micropipette. Experiments of trapping and transportation microbeads are carried out to characterize the key parameters. The results show that the trapping force can be controlled by adjusting peak-peak voltage of the sinusoidal voltage input into the piezoelectric actuator.

Comparison of AT8 immunoreactivity in the locus ceruleus and hippocampus of 154 brains from routine autopsies.

We compared semiquantitatively AT8 immunoreactivity in the locus ceruleus (LC) and hippocampus of 154 brains from routine autopsies to investigate the initial sites of phosphorylated tau (phospho-tau) development. The numbers of AT8-positive neurons and the severity of AT8-positive neuropil threads (NTs) in the LC were strongly associated: there were no cases with AT8-positive neurons that lacked NTs and 20 cases (13%) had only NTs in the LC. Phospho-tau pathologies in the LC were almost equally on both sides, although some cases (7.8%) showed unilateral predominance. The numbers of AT8-positive neurons in the LC and the numbers of AT8-positive neurons and NTs in the hippocampus were also strongly associated. There were only two cases with AT8-positive neurons in the LC that lacked phospho-tau pathology in the hippocampus, and 21 cases (13.6%) with phospho-tau pathology in the hippocampus that lacked AT8-positive neurons in the LC. The numbers of AT8-positive NTs in the LC and AT8-positive neurons and NTs in the hippocampus were also strongly associated. There were seven cases (4.5%) with AT8-positive NTs in the LC that lacked phospho-tau pathology in the hippocampus, and five cases (3.2 %) with phospho-tau pathologies in the hippocampus that lacked AT8-positive NTs in the LC. In this study, we could not confirm that phospho-tau pathologies begin in the LC. We suspect their simultaneous occurrences in both hippocampal regions and in LC.

Mechatronic Development and Vision Feedback Control of a Nanorobotics Manipulation System inside SEM for Nanodevice Assembly.

Carbon nanotubes (CNT) have been developed in recent decades for nanodevices such as nanoradios, nanogenerators, carbon nanotube field effect transistors (CNTFETs) and so on, indicating that the application of CNTs for nanoscale electronics may play a key role in the development of nanotechnology. Nanorobotics manipulation systems are a promising method for nanodevice construction and assembly. For the purpose of constructing three-dimensional CNTFETs, a nanorobotics manipulation system with 16 DOFs was developed for nanomanipulation of nanometer-scale objects inside the specimen chamber of a scanning electron microscope (SEM). Nanorobotics manipulators are assembled into four units with four DOFs (X-Y-Z-θ) individually. The rotational one is actuated by a picomotor. That means a manipulator has four DOFs including three linear motions in the X, Y, Z directions and a 360-degree rotational one (X-Y-Z-θ stage, θ is along the direction rotating with X or Y axis). Manipulators are actuated by picomotors with better than 30 nm linear resolution and <1 micro-rad rotary resolution. Four vertically installed AFM cantilevers (the axis of the cantilever tip is vertical to the axis of electronic beam of SEM) served as the end-effectors to facilitate the real-time observation of the operations. A series of kinematic derivations of these four manipulators based on the Denavit-Hartenberg (D-H) notation were established. The common working space of the end-effectors is 2.78 mm by 4.39 mm by 6 mm. The manipulation strategy and vision feedback control for multi-manipulators operating inside the SEM chamber were been discussed. Finally, application of the designed nanorobotics manipulation system by successfully testing of the pickup-and-place manipulation of an individual CNT onto four probes was described. The experimental results have shown that carbon nanotubes can be successfully picked up with this nanorobotics manipulation system.

Bending spring rate investigation of nanopipette for cell injection.

Bending of nanopipette tips during cell penetration is a major cause of cell injection failure. However, the flexural rigidity of nanopipettes is little known due to their irregular structure. In this paper, we report a quantitative method to estimate the flexural rigidity of a nanopipette by investigating its bending spring rate. First nanopipettes with a tip size of 300 nm are fabricated from various glass tubes by laser pulling followed by focused ion beam (FIB) milling. Then the bending spring rate of the nanopipettes is investigated inside a scanning electron microscope (SEM). Finally, a yeast cell penetration test is performed on these nanopipettes, which have different bending spring rates. The results show that nanopipettes with a higher bending spring rate have better cell penetration capability, which confirms that the bending spring rate may well reflect the flexural rigidity of a nanopipette. This method provides a quantitative parameter for characterizing the mechanical property of a nanopipette that can be potentially taken as a standard specification in the future. This general method can also be used to estimate other one-dimensional structures for cell injection, which will greatly benefit basic cell biology research and clinical applications.

An autopsy case of familial amyotrophic lateral sclerosis with a TARDBP Q343R mutation.

We describe a Japanese autopsy case of familial amyotrophic lateral sclerosis (FALS) with a TARDBP Q343R mutation. This male patient developed dysarthria at the age of 52 years, and bulbar symptoms progressed, with weakness and atrophy in the extremities. His mental status was normal, but he became bedridden, received artificial respiratory support at 54 years of age, and gradually acquired a locked-in state and died at 58 years of age. Microscopically, marked diffuse myelin pallor was observed in the anterolateral columns of the spinal cord. The remaining anterior horn cells contained Bunina bodies and phosphorylation-dependent transactivation response DNA-binding protein of 43 kDa (pTDP-43)-positive neuronal cytoplasmic inclusions (NCIs). Glial cytoplasmic inclusions (GCIs) were also observed. The number of ubiquitin- and p62-positive inclusions was markedly lower than that of pTDP-43-positive inclusions. NCIs and many fine dot-like pTDP-43-positive granules in the neuropil were mainly seen in the temporal and motor cortices, and striatum. NCIs were rare in hippocampal granular cells. Immunoblotting of samples from the cerebral cortex using an anti-pTDP-43 antibody was slightly different from previous TDP-43 pathological subtypes.

Cytoplasmic TDP-43 accumulation in cells of the adrenal medulla in individuals with or without amyotrophic lateral sclerosis.

The transactive response DNA-binding protein of 43 kDa (TDP-43) is normally located predominantly in the nucleus, whereas pathological TDP-43 is mostly found in the cytoplasm. Cytoplasmic TDP-43 accumulation has not yet been reported in normal general organs. In our preliminary study, paraffin sections of the general organs of individuals with or without amyotrophic lateral sclerosis (ALS) were immunostained with antibodies against TDP-43 and phosphorylated TDP-43 (pTDP-43). Diffuse and granular immunostaining pattern of TDP-43 and pTDP-43 were observed frequently in the cytoplasm of renal tubular cells, and less frequently in the cells of several organs; however, the majority of these immunoreactivities were nonspecific biotin reactions. Conversely, many TDP-43-positive and pTDP-43-negative cytoplasmic accumulations were observed in the adrenal medulla in every individual (with or without ALS). Skein-like or round inclusions were not observed. The cells in the adrenal medulla were well preserved, and the cytoplasmic TDP-43 accumulations were frequent in the cells of all routine autopsy cases without loss of nuclear TDP-43 immunostaining; therefore, we considered that this was a physiological phenomenon. This is the first study that demonstrated the cytoplasmic accumulation of TDP-43 in routinely autopsied cases.

Bioinspired Soft Spine Enables Small-Scale Robotic Rat to Conquer Challenging Environments.

For decades, it has been difficult for small-scale legged robots to conquer challenging environments. To solve this problem, we propose the introduction of a bioinspired soft spine into a small-scale legged robot. By capturing the motion mechanism of rat erector spinae muscles and vertebrae, we designed a cable-driven centrally symmetric soft spine under limited volume and integrated it into our previous robotic rat SQuRo. We called this newly updated robot SQuRo-S. Because of the coupling compliant spine bending and leg locomotion, the environmental adaptability of SQuRo-S significantly improved. We conducted a series of experiments on challenging environments to verify the performance of SQuRo-S. The results demonstrated that SQuRo-S crossed an obstacle of 1.07 body height, thereby outperforming most small-scale legged robots. Remarkably, SQuRo-S traversed a narrow space of 0.86 body width. To the best of our knowledge, SQuRo-S is the first quadruped robot of this scale that is capable of traversing a narrow space with a width smaller than its own width. Moreover, SQuRo-S demonstrated stable walking on mud-sand, pipes, and slopes (20°), and resisted strong external impact and repositioned itself in various body postures. This work provides a new paradigm for enhancing the flexibility and adaptability of small-scale legged robots with spine in challenging environments, and can be easily generalized to the design and development of legged robots with spine of different scales.

ARBUR, a machine learning-based analysis system for relating behaviors and ultrasonic vocalizations of rats.

Deciphering how different behaviors and ultrasonic vocalizations (USVs) of rats interact can yield insights into the neural basis of social interaction. However, the behavior-vocalization interplay of rats remains elusive because of the challenges of relating the two communication media in complex social contexts. Here, we propose a machine learning-based analysis system (ARBUR) that can cluster without bias both non-step (continuous) and step USVs, hierarchically detect eight types of behavior of two freely behaving rats with high accuracy, and locate the vocal rat in 3-D space. ARBUR reveals that rats communicate via distinct USVs during different behaviors. Moreover, we show that ARBUR can indicate findings that are long neglected by former manual analysis, especially regarding the non-continuous USVs during easy-to-confuse social behaviors. This work could help mechanistically understand the behavior-vocalization interplay of rats and highlights the potential of machine learning algorithms in automatic animal behavioral and acoustic analysis.

Actual conditions for returning home after hospitalization among older patients receiving home medical care in Japan: OHCARE Study.

AIM: To examine the actual conditions of older patients receiving home medical care after hospitalization over a period of 2 years in Japan. METHODS: The study population included 102 participants, aged ≥65 years, receiving home medical care, who consented to participate in the Osaka Home Care Registry (OHCARE) study in Japan over a period of 2 years. We investigated the actual conditions for returning home after hospitalization. RESULTS: The median age of the 102 participants was 84 years, and 61 (59.8%) were women. In the group that returned home, 42 (55.3%) of the respondents desired to recuperate in a familiar place, as in advanced care planning (ACP). During the 2-year follow-up period, the group that did not return home had significantly more deaths. A multivariate analysis showed the association in the presence of ACP (odds ratio: 4.72, 95% confidence interval: 1.60-13.86) and cardiac disease (odds ratio: 0.25, 95% confidence interval: 0.08-0.76). The lack of ACP in the medical records when the patient was admitted to the hospital may have prevented the return home. CONCLUSION: In older patients who had difficulty returning home after hospitalization, the lack of ACP in home medical care may have been an influencing factor. ACP could help continue with home medical care. Geriatr Gerontol Int 2024; 24: 320-326.

Single cell stiffness measurement at various humidity conditions by nanomanipulation of a nano-needle.

This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.