Abstract
In this study, we present a novel optical imaging method that makes use of high precision particle tracking of fluorescent particles to obtain images of nanometer size structures in live cells. Particle tracking not only provides the trajectory of the center of mass but also the particle orientation and size can now be observed, in vivo and real time with the nanometer resolution. This method helps in further understanding of the dynamics of the small particles in biological systems, which was hard to achieve by the current optical techniques. The method is based in rapidly modulating the position of the laser beam around small structures on the order of 100nm in size. When the laser spot oscillates in the direction toward the particle surface, the fluorescence of the particle is modulated. The modulation, which is the ratio of the alternating part to the average fluorescence intensity, is a function of the distance of the particle from the center of mass to the oscillation. In order to track the particle, we circularly moved the oscillating laser spot around the moving particle, and at the same time, analyzed the modulation in the frequency spectrum of the intensity along the orbit to perform a feedback loop updating the average laser position to the center of mass of the particle position. The size, shape and orientation information of the fluorescent structure can then be obtained by looking at the higher order modulations components. We explain the theory behind this method and we show the 3D reconstruction of nanometer microvilli structures on the apical membrane of OK cells.Work supported in part by NIH-P41 P41-RRO3155 and P50-GM076516
Published Version
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