High-Speed Focusing and Tracking of Multisized Microbiological Objects

This paper proposes a novel algorithm for high-speed autofocusing and tracking of multisized microbiological objects observed under a transmitted light microscope. Unlike well-known autofocus algorithms found in the literature, the intensity variation of only a small defined region around the border of the microobject 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 smallermicroobjects and 97 µm diameter microspheres were used to represent larger microbiological objects. The execution time and accuracy of the proposed algorithmwere assessed and better results were obtained compared to some related autofocusing algorithms. Since its computational cost was low, the algorithm facilitated highspeed autofocusing of both 3T3-SWISS cells and microspheres. The algorithm was also applied to the tracking of moving microobjects by implementing a PD controller. Since visual feedback took only about 1 ms, high-speed tracking was achieved.