Hyonchol Kim

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell.

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin beta(5) subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an independent and potentially a more quantitative method, the AFM was used with a microbead attached to the cantilever tip to increase the area of contact and VN was immobilized on the microbead. Force measurements were then performed over a large area of a living murine osteoblastic cell using the microbead covered with VN.

mRNA analysis of single living cells.

Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM). AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells.

Contribution of nanoscale curvature to number density of immobilized DNA on gold nanoparticles

We report the curvature size dependence of the density of attached single-stranded DNA (ssDNA) on the surface of gold nanoparticles. The densities of immobilized ssDNA on 10, 20, 30, and 50 nm gold nanoparticles were examined, and we found that the maximum density of the immobilized ssDNA on 10 nm particles was 13 times larger than that on 50 nm particles, which was still 10 times larger than that on flat gold surfaces. This result indicates the importance of curvature in the nanometer-scale attachment of ssDNAs to nanoparticles.

Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

Quantification of cell adhesion interactions by AFM: effects of LPS/PMA on the adhesion of C6 glioma cell to collagen type I

Abstract Time-course effects of lipopolysaccharide (LPS) with phorbol-12-myristate-13-acetate (PMA) were quantitatively examined using an atomic force microscope (AFM) cantilever with a type I collagen-coated spherical bead as a probe. LPS/PMA affects the expression level of fibronectin (FN), increasing the adhesion strength for collagen type I about 1.7 times. After 4 days, the adhesion strength for collagen increased 1.6 times. Our method is suitable for quantitative studies of cell adhesion phenomena on the single cell level.

Development of On-Chip Multi-Imaging Flow Cytometry for Identification of Imaging Biomarkers of Clustered Circulating Tumor Cells

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as “imaging biomarkers”, with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm2 and (2) nucleus area larger than 90 µm2 were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm2 were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers—cluster area, nuclei area, nuclei number, and ratio of perimeter—can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.

Cup-Shaped Superparamagnetic Hemispheres for Size-Selective Cell Filtration

We propose a new method of size separation of cells exploiting precisely size-controlled hemispherical superparamagnetic microparticles. A three-layered structure of a 2-nm nickel layer inserted between 15-nm silicon dioxide layers was formed on polystyrene cast spheres by vapor deposition. The polystyrene was then removed by burning and the hemispherical superparamagnetic microparticles, “magcups”, were obtained. The standard target cells (CCRF-CEM, 12 ± 2 μm) were mixed with a set of different sizes of the fabricated magcups, and we confirmed that the cells were captured in the magcups having cavities larger than 15 μm in diameter, and then gathered by magnetic force. The collected cells were grown in a culture medium without any damage. The results suggest that this method is quick, simple and non-invasive size separation of target cells.

DNA Hybridization Efficiency on Concave Surface Nano-Structure in Hemispherical Janus Nanocups

We examined the effect of a concave structure on DNA hybridization efficiency using an inner surface of hemispherical Janus nanocups in the range from 140 to 800 nm. Target DNA was specifically immobilized onto the inner cup surface, hybridized with complementary DNA-attached 20 nm Au probes, and the number of the hybridized probes was counted by scanning electron microscopy. The hybridization density of the attached Au probes on 800 nm nanocups was 255 μm(-2), which was 0.57 times that on a flat surface, 449 μm(-2), and increased to 394 μm(-2) on a 140 nm cup, 0.88 times of a flat surface, as the cup size decreased. The local density of attached Au probes within the central 25% at the bottom of the 800 nm nanocups was 444 μm(-2), which was closer to that on a flat surface, and the tendency was the same for all sizes of cups, indicating that the size dependency of DNA hybridization efficiency on the concave structures were mostly affected by the lower efficiency of side wall hybridization.

Production of size-controlled nanoscopic cap-shaped metal shells

A method of producing precisely size-controlled metal nanoparticles is described. Polystyrene (PS) spheres placed on a substrate were used as a cast for the metal nanoparticles. The diameters of the PS spheres were processed into the desired sizes by oxygen plasma etching, and metal was deposited on the PS to the desired thickness by thermal evaporation. The PS casts were then removed by the UV-excited ozone oxidization reaction. The diameters of the obtained cap-shaped metal shells had a distribution within 5% of the coefficient of variation. These particles can be used as simultaneously applicable biological labels along with different-sized nanoparticles in immuno-electron microscopy.

Clinical laboratory implications of single living cell mRNA analysis.

Publisher Summary The chapter presents a novel method to examine the gene expression of living individual cells using an atomic force microscope (AFM) that was used as a manipulator to extract mRNAs from cells. Relative mRNA levels of more than 500 genes change in response to stimulation. The expression pattern of each gene shows different profiles. The obtained mRNAs with AFM are analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enables to examine time-dependent gene expression of individual living cells without serious damage to the cells. Different steps involved in mRNA extraction from living cells such as preparation of cells, AFM setup, pretreatment of AFM instrument and tip, AFM operation, PCR, and expression of β-actin mRNA are also discussed in this chapter. An AFM is a powerful candidate for the manipulation of biomolecules because the AFM tip makes direct contact with the sample surface in liquid with high positional accuracy. These techniques may lead to tailor-made treatment of individual cells. Manipulations of biological material with AFM such as a protein-unfolding experiment, AFM images of subcellular structures in rat vomeronasal epithelium, the distribution of terminal GALNAc on vomeronasal epithelial sections, and a quantitative measurement of adhesion force between cell adhesion molecules and living cells with AFM are also discussed in this chapter.