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.

A tetrahedral DNA nanorobot with conformational change in response to molecular trigger.

Dynamic DNA origami nanostructures that respond to external stimuli are promising platforms for cargo delivery and nanoscale sensing. However, the low stability of such nanostructures under physiological conditions presents a major obstacle for their use in biomedical applications. This article describes a stable tetrahedral DNA nanorobot (TDN) programmed to undergo a controlled conformational change in response to epithelial cell adhesion molecule (EpCAM), a molecular biomarker specifically expressed on the circulating tumor cells. Multiresolution molecular dynamics simulations verified the overall stability of the folded TDN design and characterized local distortions in the folded structure. Atomic force microscopy and gel electrophoresis results showed that tetragonal structures are more stable than unfolded DNA origami sheets. Live cell experiments demonstrated the low cytotoxicity and target specificity of TDN. In summary, the proposed TDN can not only effectively resist nuclease catalysis but also has the potential to monitor EpCAM-positive cells precisely.

Macrophages in Giemsa-stained cerebrospinal fluid specimens predict carcinomatous meningitis.

Carcinomatous meningitis is a condition in which tumor cells spread to the subarachnoid space. Leukocyte counting and typing of cerebrospinal fluid (CSF) cell components are performed manually or using flow cytometry. However, a detailed analysis of these variables using cytological specimens has not yet been reported. The present study analyzed cytological specimens using Giemsa staining and whole slide imaging with computer-assisted image analysis (CAIA) to clarify the characteristics of the leukocyte population in CSF, especially in carcinomatous meningitis. Manual evaluation was performed using 280 Giemsa-stained cytological CSF specimens. For 49 samples, CAIA was used for the whole area of Papanicolaou (Pap) staining, and Giemsa-stained specimens of the same samples were imaged using a virtual slide scanner. The nuclear morphology of the leukocytes was assessed, and the total leukocyte and leukocyte subset (lymphocytes, neutrophils and macrophages) counts were evaluated. Then, the number and percentage of each leukocyte subset population were evaluated. The total leukocyte count was significantly higher in Giemsa-stained specimens compared with in Pap-stained specimens. The percentage of macrophages was significantly higher in samples from patients with non-hematological tumors compared with in samples from patients without tumors, which was confirmed by manual evaluation of the specimens. In addition, the cut-off value of the percentage of macrophages that could discriminate between the tumor history negative cases and cytologically tumor positive cases was determined, revealing that a higher proportion of macrophages reflected the existence of atypical/malignant epithelial tumor cells in CSF samples. Thus, atypical cell screening and analysis of the background characteristics of the leukocyte population should be the focus of cytological specimen screening, especially not to miss carcinomatous meningitis.

High neutrophil incorporation rate of ascitic fluid cytology as an indicator of cancerous ascites.

The cell­in­cell phenomenon (CiCP) involves the incorporation of a viable cell by other cells (host cells) and includes two concepts: Emperipolesis and cell cannibalism. The former involves the incorporation of hematopoietic cells as the incorporated cells, while the latter involves cell incorporation by tumor cells as host cells. A total of 239 peritoneal cavity fluid cytology specimens were evaluated for CiCP and the number of singly detectable nuclei (SDN) were measured by examining virtual slide image files. The rates of CiCP­positive cases (RCPCs) and CiCP emergence rate (CER)/SDN were significantly higher in ascites samples than in peritoneal washing samples (P<0.0001 and P=0.0026, respectively), although the numbers of SDN were not significantly different between the groups (P=0.8063). Both the RCPCs and CER/SDN were significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0220 and P=0.0312, respectively), although the numbers of SDN were not significantly different between the samples (P=0.2471). Most of the incorporated cells were lymphocytes and the host cells were macrophages; however, the rate of neutrophil incorporation (NI) by host cells in the total CiCP cells in a sample was significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0288). NI was mainly performed via emperipolesis by macrophages, with only six examples not by macrophages observed among all CiCP samples. The threshold NI rate/total CiCP (NI/CiCP) between tumor­positive and tumor­negative groups was 11.1% (P=0.0115). Using this threshold, the peripheral blood leukocyte count was significantly higher in the high­NI/CiCP group than in the low­NI/CiCP group (P=0.0022). The present findings revealed novel aspects of less frequently observed CiCP in ascitic fluid cytology by utilizing combined manual and computer assisted image analysis evaluation of samples. Notably, the present study indicated the importance of increased NI as an indicator of cancerous ascites.

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.

Magnetic Micromachine Using Nickel Nanoparticles for Propelling and Releasing in Indirect Assembly of Cell-Laden Micromodules.

Magnetic micromachines as wireless end-effectors have been widely applied for drug discovery and regenerative medicine. Yet, the magnetic assembly of arbitrarily shaped cellular microstructures with high efficiency and flexibility still remains a big challenge. Here, a novel clamp-shape micromachine using magnetic nanoparticles was developed for the indirect untethered bioassembly. With a multi-layer template, the nickel nanoparticles were mixed with polydimethylsiloxane (PDMS) for mold replication of the micromachine with a high-resolution and permeability. To actuate the micromachine with a high flexibility and large scalable operation range, a multi-pole electromagnetic system was set up to generate a three-dimensional magnetic field in a large workspace. Through designing a series of flexible translations and rotations with a velocity of 15mm/s and 3 Hz, the micromachine realized the propel-and-throw strategy to overcome the inevitable adhesion during bioassembly. The hydrogel microstructures loaded with different types of cells or the bioactive materials were effectively assembled into microtissues with reconfigurable shape and composition. The results indicate that indirect magnetic manipulation can perform an efficient and versatile bioassembly of cellular micromodules, which is promising for drug trials and modular tissue engineering.

3D Construction of Shape-Controllable Tissues through Self-Bonding of Multicellular Microcapsules.

Designed microtissues that replicate highly ordered three-dimensional (3D) multicellular in vivo structures have shown huge potential in biomedical research and drug discovery. Through microencapsulation and microfluidic techniques, cell-laden microcapsules have been widely used as pathological or pharmacological models. However, most conventional microtissue construction strategies can only engineer simply predefined microcapsules with monotonous biological components in two dimensions. Here, we propose a flexible 3D microtissue construction method through self-bonding of real-time shape-programmable microcapsules. The microcapsules are prepared by photo-induced electrodeposition of cell-laden alginate hydrogel and flexibly tailored into tissue-specific shapes, sizes, and arbitrary biocomponents. With the local fluidics-guided assembly, the microcapsules are spatially organized into 3D perfectly aligned microtissues. To mimic in vivo intercellular connection, the aligned microcapsules are precoated with fibroblasts to self-bond the adjacent layers into a robust assemblage through fibroblast-extracellular matrix interactions, which highly reproduces the tissue morphogenesis in natural organisms. As a typical complex tissue model, the 3D hepatic lobule was engineered utilizing HepG2 cells seeded into microcapsules with a fibroblast coating, and its biofunction including albumin and urea secretion was improved by nearly two-fold compared with cells seeded without a fibroblast coating. We anticipate that our method will be capable of regenerating more complex multicellular constructs with unprecedented possibilities for future tissue engineering applications.

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.

Image analysis of the nuclear characteristics of emerin protein and the correlation with nuclear grooves and intranuclear cytoplasmic inclusions in lung adenocarcinoma.

Nuclear size and shape are important components in the diagnosis of pathological specimens. However, a qualitative evaluation is typically applied rather than a quantitative evaluation technique. In the present study, we sought to evaluate the nuclear morphological characteristics of lung adenocarcinoma using whole-slide imaging (WSI) and computer-assisted image analysis (IA). We evaluated the nuclear characteristics of 106 cases of surgically resected lung adenocarcinoma according to Feulgen staining and immunohistochemistry (IHC) for the inner nuclear membrane protein emerin. According to the Feulgen reaction, although the nuclear area (size) of the carcinoma cells was correlated with the nuclear perimeter (NP) (R=0.8973), the nuclear staining intensity of carcinoma cells was not correlated with the nuclear area. Using emerin IHC, we used IA software that was able to detect both the NP and the emerin-stained nuclear membrane length (ENML) in the nucleus, and found that the more nuclei exhibited a longer ENML relative to the NP, the more nuclear grooves and intranuclear cytoplasmic inclusions were present. In addition, the nuclear area was correlated with the percentage of nuclei that had a longer ENML compared to the NP against the total nuclei (R=0.7759). Furthermore, the emerin low expression group showed an enlarged nuclear area (P=0.0264), elongated NP (P=0.0091), and lower shape factor (P=0.0486) compared with the normal emerin expression group. Our data indicated the usefulness of WSI and IA for pathological specimen analysis. In addition, this study is the first to report that the low expression of emerin in cancer cell results in an oval shape of nuclei and nuclear enlargement in clinical samples.

Fabrication of perfusable 3D hepatic lobule-like constructs through assembly of multiple cell type laden hydrogel microstructures.

The in vitro reproduction of three-dimensional (3D) cellular constructs to physiologically mimic human liver is highly desired for drug screening and clinical research. However, the fabrication of a liver-mimetic 3D model using traditional bottom-up technologies is challenging owing to the complex architecture and specific functions of real liver tissue. This work proposes a versatile strategy for spatially assembling gear-like microstructures encapsulating multiple cell types, and reorganizing them into 3D lobule-like micro-architecture with physiological relevance to native liver tissue. Gear-like microstructures were fabricated by photo-crosslinking poly(ethylene glycol) diacrylate (PEGDA) hydrogel mixed with hepatocytes and fibroblasts, in a digital micromirror device (DMD)-based microfluidic channel. The microstructures were assembled through coordinated micromanipulation based on local fluid force, and spatially self-aligned through hydrophilic-hydrophobic interactions into a 3D integrated construct with lobule-like morphology and a perfusable central lumen. The resulting 3D lobule-like constructs allowed long-term co-culture of hepatocytes and fibroblasts with high cell viability. The co-cultured constructs enhanced hepatocyte proliferation and spreading, as well as liver functions including a 50% increase in albumin secretion and urea synthesis. For hepatotoxicity assessment, the 3D lobule-like construct enabled drug perfusion through its built-in lumen for simulation of drug diffusion in the liver, which could improve the response sensitivity and efficiency to hepatotoxic drug. These results demonstrated that this method provides a valuable 3D co-culture model with perfusable lobule-like architecture and physiological functions, which has potential applications in drug discovery and tissue engineering applications.

Human papillomavirus genotyping among women with cervical abnormalities in Ulaanbaatar, Mongolia.

OBJECTIVES: Few studies on human papillomavirus (HPV) have been conducted in Mongolia. This study was performed to evaluate the prevalent HPV genotypes and their associations with cytology and demographic and behavioral characteristics in Mongolian women with cervical abnormalities. METHODS: Exfoliated cell samples of 100 women who had a previous history of cervical abnormality were collected. Cytological interpretation was conducted microscopically and HPV genotyping was performed using the Roche Linear Array test. Study questionnaires were completed. RESULTS: Overall, 25 HPV genotypes were detected in 47% of participants, and the most prevalent were HPV 16, 52, 58, and 33. Cytological examination revealed 12% of participants had atypical squamous cells of undetermined significance (ASC-US), 8% had low-grade squamous intraepithelial lesions (LSIL), 7% had high-grade squamous intraepithelial lesions (HSIL), and 14% had squamous cell carcinoma (SCC), while 59% of women had a normal cytology. HPV 16 was the most common type among women with a normal cytology and cervical cancer. However, women with cervical abnormalities including LSIL and HSIL were predominantly infected with HPV 52. Moreover, women aged <35 years had a significantly higher risk of HPV infection than those in the other age groups (p<0.05). CONCLUSIONS: The prevalent trend of HPV genotypes observed in this cohort differs from that reported previously in Mongolia. These data may contribute to developing an effective strategy for cervical cancer prevention in Mongolia.

Three-Dimensional Intravascular Reconstruction Techniques Based on Intravascular Ultrasound: A Technical Review.

Intravascular ultrasound (IVUS) imaging provides two-dimensional (2-D) real-time luminal and transmural cross-sectional images of intravascular vessels with detailed pathological information. It has offered significant advantages in terms of diagnosis and guidance and has been increasingly introduced from coronary interventions into more generalized endovascular surgery. However, IVUS itself does not provide spatial pose information for its generated images, making it difficult to construct a 3-D intravascular visualization. To address this limitation, IVUS imaging-driven 3-D intravascular reconstruction techniques have been developed. These techniques enable accurate diagnosis and quantitative measurements of intravascular diseases to facilitate optimal treatment determination. Such reconstruction extends the IVUS imaging modality from pure diagnostic assistance to intraoperative navigation and guidance and supports both therapeutic options and interventional operations. This paper presents a comprehensive survey of technological advances and recent progress on IVUS imaging-based 3-D intravascular reconstruction and its state-of-the-art applications. Limitations of existing technologies and prospects of new technologies are also discussed.

Malignant Pleural Mesothelioma with Marked Lymphatic Involvement: A Report of Two Autopsy Cases.

We herein report two cases of malignant pleural mesothelioma with marked lymphangiosis. The patients included a 68-year-old man and a 67-year-old man who both had a history of exposure to asbestos. Computed tomography (CT) on admission showed pleural effusion with pleural thickening. In both cases, a histopathological examination of the pleura confirmed the diagnosis of epithelioid malignant mesothelioma. They received chemotherapy, but the treatment was only palliative. The chest CT assessments during admission revealed marked pleural effusion and mediastinal lymphadenopathy. CT also showed a consolidative mass with bronchovascular bundle and septal thickening in the lungs suggesting pulmonary parenchymal involvement and the lymphangitic spread of the tumor. These CT findings mimicked lung cancer with pleuritis and lymphangitic carcinomatosis. Autopsy was performed in both cases. Macroscopically, the tumor cells infiltrated the lung with the marked lymphatic spread of the tumor. Microscopy also revealed that the tumor had invaded the pulmonary parenchyma with the marked lymphatic spread of the tumor. Although this growth pattern is unusual, malignant pleural mesothelioma should be considered as the differential diagnosis, especially in patients with pleural lesions.

Shape Sensing Techniques for Continuum Robots in Minimally Invasive Surgery: A Survey.

Continuum robots provide inherent structural compliance with high dexterity to access the surgical target sites along tortuous anatomical paths under constrained environments and enable to perform complex and delicate operations through small incisions in minimally invasive surgery. These advantages enable their broad applications with minimal trauma and make challenging clinical procedures possible with miniaturized instrumentation and high curvilinear access capabilities. However, their inherent deformable designs make it difficult to realize 3-D intraoperative real-time shape sensing to accurately model their shape. Solutions to this limitation can lead themselves to further develop closely associated techniques of closed-loop control, path planning, human-robot interaction, and surgical manipulation safety concerns in minimally invasive surgery. Although extensive model-based research that relies on kinematics and mechanics has been performed, accurate shape sensing of continuum robots remains challenging, particularly in cases of unknown and dynamic payloads. This survey investigates the recent advances in alternative emerging techniques for 3-D shape sensing in this field and focuses on the following categories: fiber-optic-sensor-based, electromagnetic-tracking-based, and intraoperative imaging modality-based shape-reconstruction methods. The limitations of existing technologies and prospects of new technologies are also discussed.

Pleomorphic rhabdomyosarcoma arising in the anterior mediastinum: A case report with cytological features of imprint and liquid-based cytology specimens.

We herein report the cytological features of a very rare case of pleomorphic rhabdomyosarcoma arising in the anterior mediastinum on imprint and liquid-based cytology (LBC) specimens. A 58-year-old man had an approximately 10-cm tumor in the anterior mediastinum as shown on computed tomography. Thymectomy with complete resection of the left lung was performed. The fresh cut surface of the tumor was used to prepare imprint and LBC specimens. The imprint specimens showed four types of tumor cells dispersed on a background of hemorrhage, necrosis, and mucus. On the other hand, only two types of tumor cells (spindle-shaped and spiderweb cells) were scattered or present in clusters in the LBC specimens. Immunocytologically, both of these cell types were positive for desmin and myoglobin, negative for pan-keratin and epithelial membrane antigen. Cytological and immunocytological features are useful for the correct diagnosis of pleomorphic rhabdomyosarcoma, and LBC specimens show clearer results than do imprint specimens. Diagn. Cytopathol. 2017;45:333-338. © 2016 Wiley Periodicals, Inc.

Vision-based Nano Robotic System for High-throughput Non-embedded Cell Cutting.

Cell cutting is a significant task in biology study, but the highly productive non-embedded cell cutting is still a big challenge for current techniques. This paper proposes a vision-based nano robotic system and then realizes automatic non-embedded cell cutting with this system. First, the nano robotic system is developed and integrated with a nanoknife inside an environmental scanning electron microscopy (ESEM). Then, the positions of the nanoknife and the single cell are recognized, and the distance between them is calculated dynamically based on image processing. To guarantee the positioning accuracy and the working efficiency, we propose a distance-regulated speed adapting strategy, in which the moving speed is adjusted intelligently based on the distance between the nanoknife and the target cell. The results indicate that the automatic non-embedded cutting is able to be achieved within 1-2 mins with low invasion benefiting from the high precise nanorobot system and the sharp edge of nanoknife. This research paves a way for the high-throughput cell cutting at cell's natural condition, which is expected to make significant impact on the biology studies, especially for the in-situ analysis at cellular and subcellular scale, such as cell interaction investigation, neural signal transduction and low invasive cell surgery.

Real-time in vitro intravascular reconstruction and navigation for endovascular aortic stent grafting.

BACKGROUND: Trans-catheter endovascular stent grafting minimizes trauma and increases the benefitting patient population. However, the alignment between stent graft branches and vasculature branches remains time-consuming and challenging, and such techniques require a significant amount of contrast agent for imaging. METHODS: A new framework for intravascular reconstruction based on sensor fusion between intravascular ultrasound (IVUS) imaging and electromagnetic (EM) tracking was proposed. A new image processing method was presented to realize fully automatic processing of IVUS imaging and 3D reconstruction in real time, as well as branch detection for alignment and deployment. Complementary navigation using CT data allows for efficient catheter advancement and assistant clinical judgement. RESULTS: The reconstruction of an in vitro descending aorta phantom with branches was realized at 35 Hz, with cross-section radius average error of 0.64 mm. CONCLUSION: The proposed method demonstrates significant potential for clinical applications, enables navigation for precise alignment and placement for stent grafting to reduce surgical time, and decreases hemorrhagic collisions and the use of contrast agent. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro simulator with numerical stress analysis for evaluation of stent-assisted coiling embolization in cerebral aneurysm treatments.

BACKGROUND: There are several complications associated with Stent-assisted Coil Embolization (SACE) in cerebral aneurysm treatments, due to damaging operations by surgeons and undesirable mechanical properties of stents. Therefore, it is necessary to develop an in vitro simulator that provides both training and research for evaluating the mechanical properties of stents. METHODS: A new in vitro simulator for three-dimensional digital subtraction angiography was constructed, followed by aneurysm models fabricated with new materials. Next, this platform was used to provide training and to conduct photoelastic stress analysis to evaluate the SACE technique. RESULTS: The average interaction stress increasingly varied for the two different stents. Improvements for the Maximum-Likelihood Expectation-Maximization method were developed to reconstruct cross-sections with both thickness and stress information. CONCLUSIONS: The technique presented can improve a surgeon's skills and quantify the performance of stents to improve mechanical design and classification. This method can contribute to three-dimensional stress and volume variation evaluation and assess a surgeon's skills.

A novel duct-lobular segmentectomy for breast tumors with nipple discharge using near-infrared indocyanine green fluorescence imaging.

A 44-year-old woman was referred to our hospital with pathological nipple discharge from her left breast. Ultrasonography revealed a solid tumor beneath her left areola that measured 17 mm in diameter with a dilated mammary duct. Contrast-enhanced magnetic resonance imaging showed an early-enhanced cystic tumor and a dilated mammary duct. We performed a duct-lobular segmentectomy using near-infrared indocyanine green (ICG)-fluorescence imaging. Under general anesthesia, a silicone tube was inserted into an orifice of a fluid-discharging mammary duct, and 1 mL dye-fluorescence liquid containing ICG and indigo carmine was injected into the mammary duct. A periareolar incision was made, and the fluorescence image of the demarcated mammary duct segment was obtained. The mammary duct segment was dissected, along with the demarcation line. The cystic lesion and dilated mammary duct were fully resected, and the pathological diagnosis was intraductal papilloma of the breast. We report that near-infrared ICG fluorescence could be applied for imaging of the mammary duct segment, and the fluorescence image allowed for easier duct-lobular segmentectomy for nipple discharge.

A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.