Quantitative observation of membrane-attached bio-molecules on a cell surface using gold nano-particle and atomic force microscopy

摘要

One of the technical challenges of the atomic force microscopy (AFM) is to visualize the expressed bio-molecules on a cell surface in liquid environment. A difficult point in this measurement is how to distinguish the nano-sized soft materials like specific kind of channel protein molecules on the rough cell membrane. One of the useful solution is to label such molecules using small markers like gold nano-particles attached with antibodies; however, it is still remaining a problem that the difficulty of quantifying actual sizes of nano-particles on a rough surface because of the artifact of AFM tip conformation. To overcome this problem, we have developed "curvature-reconstruction-method", in which the diameters of particles were reconstructed using the hypothesis of round shapes and fitting the trace to the arcs around the top of the particle determined from an AFM sectional image (figure 1). In detail, a fast-scan-axis line of an AFM height image containing the highest pixel was chosen and ten to twenty-percent arc of the particle was fitted with an ideal circular line to calculate diameter of the particle. By using this method instead of the measurement of particle height, 30 and 50nm of gold nano-particles were successfully distinguished on a cell surface (figure 2). For next step, we tried to identify distribution of expressed ion channel on a cell surface by applying this method in liquid. Gold nano-particles were covered with specific antibody molecules against the channel proteins and were reacted with those antigens spread on a cell surface. The labeled particles were visualized with high resolution and quantified by applying curvature-reconstruction- method in liquid environment. These results indicated that our method is one of the useful ways to study distribution of specific bio-molecules on a cell surface with high resolution.