Investigation of the Extracellular Matrix Effect for the QCM/CCD Cell Activity Monitoring System.

A real-time quartz crystal microbalance (QCM) cell activity monitoring system coupled with micro CCD cameras was developed to investigate the cultured cell activity, which could measure the viscoelastic characteristics of the cell with the QCM and observe the cell morphology changes with CCD camera simultaneously. Both the viscoelastic characteristics and the shape of the cultured cell are important factors to estimate the cell activity and the cell adhesion. The extracellular matrix (ECM) on the surface of the QCM is essential to culture the cell stably in the QCM monitoring system. To find the ECM optimization condition, the adhesive strength of cultured cells on the ECM modified glass surface was measured by using rotating water stream and CCD camera. After culturing HepG2 cells for 24 hours on the ECM modified glass plates, the glass plates were dipped in the PBS solution and rotated with 1,000, 1,300, and 1,500 rpm for 30 seconds. The adhesiveness of ECMs was investigated by calculating the remained cells after rotating. Four types of ECM, such as amino group, carboxyl group, collagen monomer, and collagen polymer, were used and tested. The current paper improves the sensing system of previous report so that measurements of four ECMs can be simultaneously conducted under the same conditions in order to enhance reliability. A collagen polymer exposed ECM was the most stable on an adhesiveness point of view, but not suitable for the QCM cell activity monitoring due to the decrease of the QCM sensitivity. The sensitivity of the QCM cell activity monitoring system using collagen monomer as ECM is about 2.6 times better than that using collagen polymer. A collagen monomer exposed ECM was more stable than amino group and carboxyl group exposed ECMs based on an adhesiveness point of view. Therefore, a collagen monomer exposed ECM was the most stable and suitable for the QCM cell activity monitoring system among the four ECMs. The changes of the resonance frequency and the resonance resistance of the ECM film with the cultured cells were investigated and compared the results of CCD camera images. From these results, we showed the QCM cell activity monitoring system coupled with the micro CCD camera could be applied to the evaluation of the cell activities.

Fabrication of a polymer-metal combined atomic force microscopy probe for coarse food surface imaging.

We fabricated a polymer-metal combined atomic force microscopy (AFM) probe by two steps; a polymeric resin was used at first step, and a metal-ion was used at second step which needs more fabricating time than the resin. At first step, we fabricated a cylindrical base on to a commercial cantilever. At second step, we fabricated a conical probe on to the fabricated cylindrical base. To make the conical probe composed with silver, a 0.2 M aqueous solution of silver nitrate (AgNO3) was used. A 50 microm length polymeric-metallic hybrid tip has been fabricated to observe large bio and food samples. Generally, the AFM images of bio/food samples show cliff-like sharp patters in vertical. However, the AFM image by fabricated long tip shows clear structure of each brown rice flours. As most of commercial tips have three-angular pyramidal, the scanned results should be influenced by the lateral face of the three-angular pyramid, which results in cliff-like images. Because the sample size is large, the side area of the sample was adversely affected by the pyramidal structure during imaging. This problem may be resolved by designing conical structure tips. As the conical structure has no edge, the AFM image becomes clear. The fabricated tip has conical structure, and a clear AFM image was achieved.

Observation of interaction force in large area between actin modified surface and actin antibody modified microsphere probe.

In order to observe the force interaction in large areas, a novel force detection probe was fabricated by two-photon absorbed photopolymerization (TPAP) techniques. The probe was based on a commercial cantilever, and a docking structure for adopting a microsphere immobilized with actin antibody was fabricated by the TPAP techniques. The commercial AFM tip was also modified with the antibody for comparison. Using force curve measurement, the interaction force was compared between the modified probes and the sample surface which was immobilized with actin using a spotting system. The adhesive force of 1.3 nN was measured applying the commercial cantilever. The value was comparable to the measured interaction force of 130 nN applying the microsphere modified cantilever. The measured adhesive force of the novel probe was 100-fold larger than that obtained by the sharp AFM cantilever tip. This strong adhesive force of the microsphere modified cantilever to actin is explainable by the large contact area between the microsphere and the sample surface.

Increase the reliability and applicability of quartz crystal resonator coupled with other instruments.

The principle and applications of quartz-crystal resonator systems and the methods to increase their reliability and applicability coupled with other instruments were reported. The principle of quartz-crystal resonator system was documented with based on the three basic concepts for mass, viscosity, and viscoelastic changes. In the preliminary discussion, the realization of a resonant frequency-resonant resistance diagram was described in detail. As the examples of quartz-crystal resonator applications with introducing the resonant resistance concept and the resonant frequency, the fabrication of a carbon-coated quartz crystal sensor and monitoring changes in the viscoelastic properties of thin polymer films were carefully discussed. Examples for increasing their reliability and applicability of quartz crystal resonator systems combined with UV-visible spectroscopy, Atomic Force Microscope (AFM), Surface Plasmon Resonance (SPR), or Charge Coupled Device (CdCD) camera were described.

Influence of diquat on growth and death of HepG2 cells using quartz crystal and micro CCD camera.

Diquat is widely used agent which produces toxicity in human and implicated as an environmental toxicity. HepG2 cell was cultured onto an indium tin oxide (ITO) surface of quartz crystal modified a collagen film. In this paper, we investigated the physical properties and the morphological change of the HepG2 cells cultured onto the ITO electrode of the quartz crystal sensor with micro CCD camera. The resonance responses of the quartz crystal and the morphological change were directly monitored. After seeding the cells and diquat injection into the chamber, the resonance frequency and the resonance resistance were obtained with real time morphologies. From the resonance characteristics and the series of morphologies, we could know the diquat to be death and weakening of the cells.

Optical and electrical properties of merocyanine dye LB films by various time of UV irradiation and heat treatment.

The Langmuir-Blodgett (LB) technique provides many possibilities for the control of film thickness, dimensions, and molecular structures on the nanometer scale. Various kinds of dye molecules have been found to form the J-aggregation which has been used as sensitizers of silver halide photography for long time. In recent years, they attract attention as model systems for investigating the ultra-fast exciton dynamics, materials for ultra-fast nonlinear optical devices, fluorescence probes for mitochondrial membranes. We fabricated the merocyanine dye LB films with arachidic acid (AA). In order to observe the J-aggregation of the merocyanine dye LB films, CdCl2 and KHCO3 solutions were added in subphase. From the optical absorption spectra of the mixed dye LB films (6Me-Ds:AA = 1:2) at different layers, the optical absorption peak was about 520 nm. However, the optical absorption peak of the LB films was shifted to 600 nm, when CdCl2 and KHCO3 solutions were added. This is the consequence result to the J-aggregation of the merocyanine dye. We also investigated the optical absorption peak of the LB films according to various time at 60 degrees C and 275 nm UV. We measured the STM morphology of the merocyanine dye LB film (1 layer) before UV irradiation and heat treatment. The morphology size of the LB film on HOPG was 5 nm. The roughness and molecular size were about 66.163 pm and 0.176 nm, respectively. The J-aggregation of this type was also accompanied by large morphological changes. We analyze the morphology and electrical properties of the LB films by the scanning tunneling microscopy (STM).

Monitoring of anticancer effect of cisplatin and 5-fluorouracil on HepG2 cells by quartz crystal microbalance and micro CCD camera.

We have developed a monitoring system for evaluating the effect of anticancer agents, such as cisplatin and 5-fluorouracil (5-FU). The system mainly consisted of a quartz crystal microbalance (QCM) with indium tin oxide (ITO) electrodes and a micro CCD camera that can function in a humid CO(2) incubator. Human hepatoma cell line HepG2 cells were cultured and treated with the anticancer agents. As the behavior on the resonance frequency (F)-resonance resistance (R) diagram shows the viscoelastic change on the surface of the QCM, the effect of the anticancer agent was evaluated with the F-R diagram and the micro CCD camera, in comparison with the results in the case of general culturing (no anticancer agent injection). During general culturing, the resonance frequency decreased and the resonance resistance increased. This means that the mass loading of a viscous material occurred on the QCM. Observing with the micro CCD camera, the cancer cells were spread, divided, and the number of the cells increased. On the other hand, when the anticancer agent was injected to the culturing cancer cells, the resonance frequency and the resonance resistance increased continuously. This means a decrement of the mass effect and an increment of the viscosity on the QCM. From the observation with the micro CCD camera, the number of the cells did not change. The cells shrinked and changed the shape flat to round by loosing the cell activity in the case of 5-FU treatment. These results indicate the anticancer agents were effective to the culturing cancer cells.

Monitoring of cultured cell activity by the quartz crystal and the micro CCD camera under chemical stressors.

For investigating effects of chemical stressors to cultured cells, we have developed a quartz crystal microbalance (QCM) system with a micro CCD camera that enables microscopic observations simultaneously with the QCM measurements. Human hepatoma cell line (HepG2) cells were cultured on the collagen coated quartz crystal which has indium tin oxide (ITO) electrodes that enable transmission imaging of the cultured cells by the micro CCD camera during the QCM measurements. Glutaraldehyde (GA) and t-butylhydroperoxide (t-BHP) were used for the chemical stressors. The response of the QCM was monitored and analyzed with the resonance frequency and the resonance resistance (F-R) diagram. At the same time, the photographs of the cells were recorded to observe the morphological change. In the case of GA, the QCM responded in two steps which consisted of the rapid response of the cross-linking reactions and successive decreasing cytoskeletons in the cells. In the case of t-BHP, the response showed two steps. At first, the cells changed their shapes to round, and then the weakened cells were unsticked from the surface.

Monitoring of morphology and physical properties of cultured cells using a micro camera and a quartz crystal with transparent indium tin oxide electrodes after injections of glutaraldehyde and trypsin.

For investigating the effects of chemical stimulation to cultured cells, we have developed a quartz crystal sensor system with a micro charge-coupled device (CCD) camera that enables microphotograph imaging simultaneously with quartz crystal measurement. Human hepatoma cell line (HepG2) cells were cultured on the quartz crystal through a collagen film. The electrode of the quartz crystal was made of indium tin oxide (ITO) transparent electrodes that enable to obtain a transparent mode photograph. Glutaraldehyde and trypsin were injected to the chamber of the cells, respectively. The response of the quartz crystal was monitored and microphotographs were recorded, and the resonance frequency and resonance resistance were analyzed with an F-R diagram that plotted the resonance frequency and resonance resistance. In the case of the glutaraldehyde injection, the cells responded in two steps that included the fast response of the cross-linking reaction and the successive internal change in the cells. In the case of the trypsin injection, the responses included two processes. In the first step, cell adhesion factors were cleaved and the cell structure became round, and in the next step, the cells were deposited on the quartz crystal surface and the surface of the cells was directly in contact with the quartz crystal surface.