High-Speed Auto Focusing - Depth From Diffraction (DFDi)

Concept

Automated microscopic measurement of biological specimens is becoming increasingly important in medicine and life sciences. A critical step in such measurement is autofocusing. Since the depth of focus of microscopes is very shallow, on the order of several micrometers, small shifts in the depth direction cause the specimen to easily become out of focus; thus, autofocusing is essential to keep the object in focus for precise observation. Furthermore, major applications of such automated measurement, such as image cytometry, require high-throughput because the number of the target specimens tends to be enormous. Therefore, high-speed operation is also important.

Many microscope focusing methods based on the spatial frequency of the acquired image have been proposed. The best focal position providing the highest amount of detail can be estimated from a focus curve, formed by sampling a focus score and plotting it against focal position in the depth direction. The best focal position is then found by searching for the peak in the focus curve. However, sampling of the focus curve takes a considerable amount of time, because many images at many focal positions must be individually acquired and processed.

To solve this problem, we proposed a new autofocusing method for microbiological specimens, such as cells. The proposed focusing method used a quick focus estimation named ``depth from diffraction'', based on a diffraction pattern in a defocused image of a biological specimen. Since this method can estimate the focal position of the specimen from only a si ngle defocused image, it can easily realize high-speed autofocusing.

For example, here shows the diffraction patterns generated by the target specimen, a paramecium. Bright and dark fringe position changes depending on the z-position of the specimen.

References

1. Theodorus, Hiromasa Oku, Koichi Hashimoto and Masatoshi Ishikawa. Optical axis tracking of microorganism using high speed vision. Focus on Microscopy 2005 (Jena, Gernamy, 2005.3.22) / Program and Abstract Book, pp. 105.