High-speed focusing and tracking of multisized microbiological objects

This paper proposes a novel algorithm for high-speed auto focusing and tracking of multi sized microbiological objects observed under a transmitted light microscope. Unlike well-known auto focus algorithms found in the literature, the intensity variation of only a small defined region around the border of the micro object is analyzed in the frequency domain to determine the focused position of the object quickly. In the experiment, 20 μm3T3-SWISS cells were used as smaller micro objects and 97 μm diameter micro spheres were used to represent larger microbiological objects. The execution time and accuracy of the proposed algorithm were assessed and better results were obtained compared to some related auto focusing algorithms. Since its computational cost was low, the algorithm facilitated high speed auto focusing of both 3T3-SWISS cells and micro spheres. The algorithm was also applied to the tracking of moving micro objects by implementing a PD controller. Since visual feedback took only about 1 ms, high-speed tracking was achieved.