Lianqing Liu

Sensor Referenced Real-Time Videolization of Atomic Force Microscopy for Nanomanipulations

The main problem of atomic force microscopy (AFM)-based nanomanipulation is the lack of real-time visual feedback. Although this problem has been partially solved by virtual reality technology, the faulty display caused by random drift and modeling errors in the virtual reality interface are still limiting the efficiency of the AFM-based nanomanipulation. Random drift aroused from an uncontrolled manipulation environment generates a position error between the manipulation coordinate and the true environment. Modeling errors due to the uncertainties of the nanoenvironment often result in displaying a wrong position of the object. Since there is no feedback to check the validity of the display, the faulty display cannot be detected in real time and leads to a failed manipulation. In this paper, a real-time fault detection and correction (RFDC) method is proposed to solve these problems by using the AFM tip as an end effector as well as a force sensor during manipulation. Based on the interaction force measured from the AFM tip, the validity of the visual feedback is monitored in real time by the developed Kalman filter. Once the faulty display is detected, it can be corrected online through a quick local scan without interrupting manipulation. In this way, the visual feedback keeps consistent with the true environment changes during manipulation, which makes it possible for several operations to finish without an image scan in between. The theoretical study and the implementation of the RFDC method are elaborated. Experiments of manipulating nanomaterials including nanoparticles and nanorods have been carried out to demonstrate its effectiveness and efficiency.

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt’s lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young’s modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young’s modulus than that of indolent cancer cells, which may improve our understanding of metastasis.

Atomic force microscopy study of the antigen-antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright

AFM-Based Robotic Nano-Hand for Stable Manipulation at Nanoscale

One of the major limitations for Atomic Force Microscopy (AFM)-based nanomanipulation is that AFM only has one sharp tip as the end-effector, and can only apply a point force to the nanoobject, which makes it extremely difficult to achieve a stable manipulation. For example, the AFM tip tends to slip-away during nanoparticle manipulation due to its small touch area, and there is no available strategy to manipulate a nanorod in a constant posture with a single tip since the applied point force can make the nanorod rotate more easily. In this paper, a robotic nano-hand method is proposed to solve these problems. The basic idea is using a single tip to mimic the manipulation effect that multi-AFM tip can achieve through the planned high speed sequential tip pushing. The theoretical behavior models of nanoparticle and nanorod are developed, based on which the moving speed and trajectory of the AFM tip are planned artfully to form a nano-hand. In this way, the slip-away problem during nanoparticle manipulation can be get rid of efficiently, and a posture constant manipulation for nanorod can be achieved. The simulation and experimental results demonstrate the effectiveness and advantages of the proposed method.

Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ∼200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

Nanoscale monitoring of drug actions on cell membrane using atomic force microscopy

Knowledge of the nanoscale changes that take place in individual cells in response to a drug is useful for understanding the drug action. However, due to the lack of adequate techniques, such knowledge was scarce until the advent of atomic force microscopy (AFM), which is a multifunctional tool for investigating cellular behavior with nanometer resolution under near-physiological conditions. In the past decade, researchers have applied AFM to monitor the morphological and mechanical dynamics of individual cells following drug stimulation, yielding considerable novel insight into how the drug molecules affect an individual cell at the nanoscale. In this article we summarize the representative applications of AFM in characterization of drug actions on cell membrane, including topographic imaging, elasticity measurements, molecular interaction quantification, native membrane protein imaging and manipulation, etc. The challenges that are hampering the further development of AFM for studies of cellular activities are aslo discussed.

Three-Dimensional Super-Resolution Morphology by Near-Field Assisted White-Light Interferometry

Recent developments in far-field fluorescent microscopy have enabled nanoscale imaging of biological entities by ingenious applications of fluorescent probes. For non-fluorescence applications, however, scanning probe microscopy still remains one of the most commonly used methods to “image” nanoscale features in all three dimensions, despite its limited throughput and invasiveness to scanned samples. Here, we propose a time-efficient three-dimensional super-resolution microscopy method: near-field assisted white light interferometry (NFWLI). This method takes advantage of topography acquisition using white-light interferometry and lateral near-field imaging via a microsphere superlens. The ability to discern structures in central processing units (CPUs) with minimum feature sizes of approximately 50 nm in the lateral dimensions and approximately 10 nm in the axial dimension within 25 s (40 times faster than atomic force microscopes) was demonstrated. We elaborate in this paper the principles of NFWLI and demonstrate its potential for becoming a practical method for high-speed and non-toxic three-dimensional nanoscale imaging.

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkins lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500 × 500 nm(2)) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells.

Cutting forces related with lattice orientations of graphene using an atomic force microscopy based nanorobot

The relationship between cutting forces and lattice orientations of monolayer graphene is investigated by using an atomic force microscopy (AFM) based nanorobot. In the beginning, the atomic resolution image of the graphene lattice is obtained by using an AFM. Then, graphene cutting experiments are performed with sample rotation method, which gets rid of the tip effect completely. The experimental results show that the cutting force along the armchair orientation is larger than the force along the zigzag orientation, and the cutting forces are almost identical every 60 degrees, which corresponds well with the 60 degrees symmetry in graphene honeycomb lattice structure. By using Poisson analysis method, the single cutting force along zigzag orientation is 3.9 nN, and the force along armchair is 20.5 nN. This work lays the experimental foundation to build a close-loop fabrication strategy with real-time force as a feedback sensor to control the cutting direction