High-Speed Scanning Microscope by Depth From Diffraction (DFDi) Method

Summary

When the number of specimens is large, it is impossible to observe all specimens in a static field of view of a microscope due to the limited resolution of the microscope or camera. A scanning microscope solves this problem by moving its field of view to observe the specimen as a sequence of images. Scanning microscopes are commonly used in the field of cytometry.

Autofocusing is essential for the scanning microscope to obtain a precise image of the specimen. It is not possible to maintain focus simply by determining the best focus depth at two points on a microscope slide and scanning along the line between them in three-dimensional space. There may be many reasons for this, including mechanical instability of the microscope and irregularity of the glass slide surface.

Furthermore, major applications of such automated measurement, such as image cytometry, require high throughput because the number of target specimens tends to be enormous. Therefore, high-speed autofocusing is important.

We developed a high-speed scanning microscope using high-speed autofocusing algorithm based on Depth From Diffraction (DFDi) method and DFDi algorithm for multiple cells that we've developed. High-speed scanning and observation of enormous yeast cells were conducted to confirm it's validity. Results are shown below. Left image sequence are captured images while laterally scanning the specimens. At first (a), the incoming two cells were out of focus. As they entered inside of the field of view (b), the system started autofocusing and move them into focus just in 40 ms (f). Right plot shows depth position of the target. Videos are also available below in this page.

Movies

References

1. Hiromasa Oku, Soshiro Makise, Masatoshi Ishikawa : High-Speed Autofocusing of Cells Using Radial Intensity Profiles Based on Depth From Diffraction (DFDi) Method, Journal of Aero Aqua Bio-mechanisms, Vol.3, No.1, pp.13-21 (2013) [Doi:10.5226/jabmech.3.13]
2. Soshiro Makise, Hiromasa Oku, and Masatoshi Ishikawa : Serial Algorithm for High-speed Autofocusing of Cells using Depth From Diffraction (DFDi) Method, 2008 IEEE International Conference on Robotics and Automation (ICRA 2008) (Pasadena, 2008.5.23)/Conference Proceedings, pp.3124-3129 [PDF (4.1M)] ^*IEEE
3. Hiromasa Oku, Masatoshi Ishikawa, Theodorus, and Koichi Hashimoto : High-speed autofocusing of a cell using diffraction pattern, Optics Express 14, pp.3952-3960 (2006) http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-9-3952

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