[en] 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. (paper)

[en] The fabrication of alginate hydrogel in 3D has recently received increasing attention owing to its distinct efficacy as biocompatible scaffold for 3D cell culture, biomedical and tissue engineering. We report a controllable 3D alginate hydrogel patterning method by developing a visible-light induced electrodeposition chip. The chip mainly consists of a photoconductive titanyl phthalocyanine (TiOPc) anode plate, an indium tin oxide (ITO) cathode plate and the mixed solution (1% sodium alginate and 0.25% CaCO₃ nano particles) between them. After a designed visible-light pattern is projected onto the TiOPc plate, the produced H⁺ by electrolysis will trigger Ca²⁺ near the anode (illuminated area), and then the gelation of calcium alginate patterns, as desired, happens controllably. In addition, we further establish an exponential model to elucidate the gel growth v.s. time and current density. The results indicate that the proposed method is able to fabricate various 3D alginate hydrogel patterns in a well controllable manner, and maintain the laden cells at high survival rate (>98% right after gel formation). This research paves an alternative way for 3D alginate hydrogel patterning with high controllability and productivity, which would benefit the research in biomedical and tissue engineering. (paper)

[en] Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level

[en] 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. (paper)

[en] A novel nanoknife with a buffering beam is proposed for single-cell cutting. The nanoknife was fabricated from a commercial atomic force microscopy (AFM) cantilever by focused-ion-beam (FIB) etching technique. The material identification of the nanoknife was determined using the energy dispersion spectrometry (EDS) method. It demonstrated that the gallium ion pollution of the nanoknife can be ignored during the etching processes. The buffering beam was used to measure the cutting force based on its deformation. The spring constant of the beam was calibrated based on a referenced cantilever by using a nanomanipulation approach. The tip of the nanoknife was designed with a small edge angle 5 deg. to reduce the compression to the cell during the cutting procedure. For comparison, two other nanoknives with different edge angles, i.e. 25 deg. and 45 deg., were also prepared. An in situ single-cell cutting experiment was performed using these three nanoknives inside an environmental scanning electron microscope (ESEM). The cutting force and the sample slice angle for each nanoknife were evaluated. It showed the compression to the cell can be reduced when using the nanoknife with a small edge angle 5 deg. Consequently, the nanoknife was capable for in situ single-cell cutting tasks.

[en] Glass nanoneedles are important tools for injecting drugs and other materials into living cells. Although we know a great deal about the mechanical properties of glass structures at the millimeter scale, relatively little is known at the nanoscale. Here we investigate the mechanical performance of hollow glass nanoneedles by nanorobotic in situ manipulation inside SEM. Quartz and borosilicate nanoneedles fabricated from glass capillaries are assembled on the nanorobotic characterization system inside SEM and their behaviors during bending and recovery are studied in situ . The result indicates the glass nanoneedle could present a large elastic bending deformation (>90°). Specifically, the quartz nanoneedle takes on larger bending strength and its deformation can recover totally. In contrast, the borosilicate nanoneedle presents more flexible and still 20% of deformation is remained after 3 months. These results not only enhances our basic understanding on nanoglass materials but also provides references for practical nanomanipulation applications. (paper)

[en] Highlights: → A nano-picker is developed for single cell adhesion force measurement. → The adhesion of picker-cell has no influence to the cell-cell measurement result. → Cell-cell adhesion force has a rise at the first few minutes and then becomes constant. -- Abstract: Cell's adhesion is important to cell's interaction and activates. In this paper, a novel method for cell-cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell-cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell-cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell-cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially.

[en] Calcium alginate hydrogels are widely used as biocompatible materials in a substantial number of biomedical applications. This paper reports on a hybrid 3D printing and electrodeposition approach for forming 3D calcium alginate hydrogels in a controllable manner. Firstly, a specific 3D hydrogel printing system is developed by integrating a customized ejection syringe with a conventional 3D printer. Then, a mixed solution of sodium alginate and CaCO₃ nanoparticles is filled into the syringe and can be continuously ejected out of the syringe nozzle onto a conductive substrate. When applying a DC voltage (∼5 V) between the substrate (anode) and the nozzle (cathode), the Ca²⁺ released from the CaCO₃ particles can crosslink the alginate to form calcium alginate hydrogel on the substrate. To elucidate the gel formation mechanism and better control the gel growth, we can further establish and verify a gel growth model by considering several key parameters, i.e., applied voltage and deposition time. The experimental results indicate that the alginate hydrogel of various 3D structures can be formed by controlling the movement of the 3D printer. A cell viability test is conducted and shows that the encapsulated cells in the gel can maintain a high survival rate (∼99% right after gel formation). This research establishes a reliable method for the controllable formation of 3D calcium alginate hydrogel, exhibiting great potential for use in basic biology and applied biomedical engineering. (paper)

[en] Highlights: • Developing a 360° multiparametric imaging atomic force microscopy based on micro/nano robot technique. • Proposing a 3D reconstruction method of the small scale sample’s topography 5 and nanomechanical properties. • Integrating a three degrees of freedom high-precision rotation stage and proposing a home positioning approach to compensate for the eccentric distance. • Performing 360° multiparametric mapping and 3D reconstruction (e.g., topography, adhesion, modulus, energy dissipation) of a human hair sample. -- Abstract: Atomic Force Microscopy (AFM) has been intensively used for imaging, characterization and manipulation at the micro- and nanoscale. Taking into account that the material is usually anisotropic, it needs to be characterized in various regions and orientations. Although recent advances of AFM techniques have allowed for large area scan of the sample on a two-dimensional plane, mapping a three-dimensional (3D) sample at a full orientation of 360° remains challenge. This paper reports a multiparametric imaging atomic force microscope via robot technique for 360° mapping and 3D reconstruction of the sample’s topography and nanomechanical properties. The system is developed by integrating a three degrees of freedom (DoFs) high-precision rotation stage and a home positioning approach is proposed to compensate for the eccentric distance between the cross-section center of the sample and the ration center of the stage. With this method, the sample surface can be fully mapped by the force-distance-based AFM via rotating the sample with a complete orientation. 360° multiparametric mapping and 3D reconstruction results (e.g., topography, adhesion, modulus, energy dissipation) of a human hair demonstrate practicability and reliability of the proposed method.

[en] A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research. -- Research highlights: → A nanorobotic manipulation system was developed inside an ESEM. → A micro-puller was designed for single yeast cell adhesion force measurement. → Yeast cells were still alive after being observed and manipulated inside ESEM. → Yeast cell adhesion force to substrate is smaller at high humidity condition than at low humidity condition.