High-performance autofocus circuit for biological microscopy

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Abstract
An autofocus circuit, based on measurement of the high spatial frequency image components, was designed for automated microscopic scanning of biological specimens. By careful consideration of the system transfer function, elimination of video end-of-line filter artifacts, correction for illumination instability, and incorporation of autogain, the focus measurement circuit attained the sensitivity and dynamic range necessary for robust operation even at the extremes of biological specimen detail encountered in exhaustive raster scans of thousands of fields. This new circuit exhibited a 25-fold improvement in dynamic range over a previous analog implementation, matched real-time digital performance at an order of magnitude lower cost, resulted in autofocus precision of 56 nm (or 10-fold better than the depth of field of the objective) in scanning experiments comprising over 10 000 microscope fields, and tracked focus at scanning speeds of up to 3.45 fields/s. Focus was correctly maintained in these scanning experiments without additional compensation for low-detail images. This circuit makes possible the widespread inclusion of high-performance autofocus as a low cost option in video microscopy systems.

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