| Single particle tracking technology is one of the most important research tools for many biological investigations such as single-molecule dynamics. To obtain the folding and unfolding information of the biological macromolecule, precise three-dimensional position measurement of the micrometer scale spheres with nanometer even sub-nanometer resolution is needed. In recent years, while 1nm resolution location in lateral plane is achieved by Hough transform etc., the high resolution measurement in the axial direction, of which a 10 nm order resolution is realized with difficulty, is always the bottleneck when locating the three-dimensional position of microspheres. The characteristics of digital in-line holographic microscopy being able to measure the overlapping spheres in axial direction and off-focus imaging being capable to measure the axial position of single microsphere with particularly high resolution, has made them the focus followed by a large number of researchers all over the world. But there still exists some technical barriers waiting to be resolved promptly.To meet the demand of high resolution measurement in the axial direction, the method of off-focus imaging based on cross-correlation or radius projecting and the method of digital in-line holographic microscopy are discussed in this paper. By research sequence, the work in the thesis can be listed as the following:1. The measurement methods of microspheres in the axial direction based on both theoretical algorithm fitting and experiment images fitting are discussed and summarized carefully, including the measuring principle, the characteristics of the object, the system, the resolution and so on. It is concluded that the digital in-line holographic microscopy and off-focus imaging would be the most effective methods of particle tracking in the near future.2. Holographic deconvolution microscopy is put forward to measure the axial position of the microsphere, in which interpolation and fitting methods are induced during and after the reconstruction progress. In this case, a resolution of 2nm for a single microsphere tracking is achieved and a better than 5nm resolution for the overlapping spheres is also realized.3. The experimental test is executed by off-focus imaging methods based on cross-correlation and radius projecting. As a result, the experiments show that a standard deviation is 0.64 nm was realized when tracking the same microsphere continuously in the axial direction in 4 minutes, and a highest of 1nm resolution is achieved. Comparative measurement with the digital in-line holographic microscopy under the same experimental conditions is also finished. |
