Takaharu Okajima

Kinetics of volume phase transition in poly(N-isopropylacrylamide) gels

Kinetics of volume phase transition in poly(N-isopropylacrylamide) (NIPA) gels jumped from a low-temperature swollen phase to a high-temperature shrunken phase was studied as functions of NIPA monomer and crosslinker concentrations. We found for the first time a clear kinematical boundary at which the shrinking relaxation time of gels changes discontinuously by 102–104 times, and that the profile of the boundary correlates with the sol-gel transition line and the contour line of turbidity of gels. A “morphological” boundary which characterizes the emergence of the bubble formation on gel surface was also determined. The theoretical calculation of the phase diagram on the basis of the mean field theory shows qualitatively that the shrinking speed of gels could be connected with the depth of the thermodynamic region of the spinodal instability (K+4μ/3=0) into which they are transferred where K and μ are the bulk and the shear moduli, respectively. A mechanism of discontinuous change of the shrinking speed i...

Self-oscillation technique for AFM in liquids

Abstract A simple self-oscillation circuit was built into a commercially available atomic force microscope (AFM) apparatus and used for imaging sample surfaces with a high rigidity in liquid environments. Imaging experiments were conducted with an AFM tip recommended for the normal tapping (intermittent contact) mode in liquids. In spite of the existence of many artificial resonance peaks, the self-oscillation could be successfully achieved with our simple electronic circuits. Force curves obtained in liquids showed that, as the tip contacted the surface, the resonance frequency began to increase steeply without a clear reduction of the vibration amplitude. Image of the sample surface was stably obtained under the condition of the positive frequency shift within a few Hertzs, which corresponded to a contact force comparable with Q -control operation.

Study of shear force between glass microprobe and mica surface under controlled humidity

Resonance characteristics of a glass micropipette near a mica surface have been investigated as functions of the tip–surface distance, D, and of the ambient humidity, H. With decreasing D, the resonance frequency of the tip increases, while its resonance amplitude decreases. The resonance curve is almost symmetric except in the close vicinity of D=0, at which the oscillation is damped completely. The effective length, D0, of the tip–surface interaction is independent of the dither amplitude. With increasing H, D0 decreases gradually, exhibiting a rather sharp drop around H=40%. These results indicate that, for large D where the direct contact does not occur, some noncontact force between the tip and the surface is operative, and that this force is sensitive to the presence of a water layer on the surface.

Nano-Mechanical Methods in Biochemistry using Atomic Force Microscopy

The atomic force microscope has been extensively used not only to image nanometer-sized biological samples but also to measure their mechanical properties by using the force curve mode of the instrument. When the analysis based on the Hertz model of indentation is applied to the approach part of the force curve, one obtains information on the stiffness of the sample in terms of Youngs modulus. Mapping of local stiffness over a single living cell is possible by this method. The retraction part of the force curve provides information on the adhesive interaction between the sample and the AFM tip. It is possible to functionalize the AFM tip with specific ligands so that one can target the adhesive interaction to specific pairs of ligands and receptors. The presence of specific receptors on the living cell surface has been mapped by this method. The force to break the co-operative 3D structure of globular proteins or to separate a double stranded DNA into single strands has been measured. Extension of the method for harvesting functional molecules from the cytosol or the cell surface for biochemical analysis has been reported. There is a need for the development of biochemical nano-analysis based on AFM technology.

Use of AFM for imaging and measurement of the mechanical properties of light-convertible organelles in plants.

We obtained topographic images of etioplasts and chloroplasts and measured their elasticity in a physiological buffer using an atomic force microscope (AFM) and found a possible correlation between the morphological and mechanical properties during the light conversion of etioplasts to chloroplasts. Alcian blue 8GX dye was found to be effective for immobilizing the plant organelles stably on a glass surface for AFM experiments. We employed the tapping-mode AFM with a cantilever soft enough to measure the elasticity of the organelles in a liquid solution. The best images of soft, spherical organelles were obtained using the tapping-mode AFM with oscillation at the thermal vibration frequency of the cantilever of around 3 kHz. Whereas etioplasts were found to be smooth-surfaced and stiff against compression by the AFM tip, before light conversion to chloroplasts, they became rough-surfaced and mechanically soft after exposure to light. The elasticity of etioplasts was 20 times higher than that of chloroplasts, probably reflecting changes in their inner structures.

Frequency shift feedback imaging in liquid for biological molecules

Abstract A commercially available atomic force microscope (AFM) equipped with a hand made simple self-oscillation circuit was used in imaging biomolecular samples in liquid environments, i.e. under physiological conditions. Assembled tau proteins, which are the major component of the neurofibrillary deposits in Alzheimer’s disease, was taken as a trial sample. In order to image its native structure, the protein was physically absorbed on a cleaved mica surface without fixation. Using the frequency feedback imaging with a self-oscillation technique, the structure of protein fibers was clearly imaged even in a wide scanning range (3.75xa0μm) with a contact force less than 100xa0pN. Furthermore, no damage of the proteins was observed in successive imagings. This indicates that the deformation of proteins was negligible in our method. In contrast, the proteins were destroyed when the vertical applied force of above 300xa0pN was applied using the amplitude feedback imaging with the self-oscillation technique.

Non-destructive force measurement in liquid using atomic force microscope

Abstract Atomic force microscopy (AFM) has been applied to measure inter- or intra-molecular forces acting to hold biological molecules and structures. For these measurements, it is important to keep the target molecules biologically active on a solid surface. Besides the strategy for immobilizing them on the surface keeping their biological activities intact, it is crucial to reduce the force applied to them through the AFM tip to avoid mechanical inactivation of the sample. In this paper, we propose a new procedure to minimize the effect of contact force. The first step of the procedure is to bring the cantilever tip close to the sample surface within less than 3xa0μm, but short of contact with the sample surface. The approximate distance of the tip from the sample stage is measured using the thermal fluctuation of the cantilever. The second step is a “compression-free” force spectroscopy for the measurement of protein–protein interactions only, which is possible when the piezo scanner was retracted before the cantilever starts upward deflection. The interaction force can be measured in the retraction period provided a physical contact is established between the proteins on the tip and the substrate. This procedure allowed to measure interaction forces between GroEL and a denatured protein without mechanical deformation.