HIGH-PERFORMANCE AUTOFOCUS CIRCUIT FOR BIOLOGICAL MICROSCOPY

An autofocus circuit, based on measurement of the high spatial frequen cy image components, was designed for automated microscopic scanning o f biological specimens. By careful consideration of the system transfe r function, elimination of video end-of-line filter artifacts, correct ion for illumination instability, and incorporation of autogain, the f ocus measurement circuit attained the sensitivity and dynamic range ne cessary for robust operation even at the extremes of biological specim en detail encountered in exhaustive raster scans of thousands of field s. This new circuit exhibited a 25-fold improvement in dynamic range o ver a previous analog implementation, matched real-time digital perfor mance at an order of magnitude lower cost, resulted in autofocus preci sion of 56 nm (or 10-fold better than the depth of field of the object ive) in scanning experiments comprising over 10000 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 w idespread inclusion of high-performance autofocus as a low cost option in video microscopy systems. (C) 1998 American Institute of Physics. [S0034-6748(98)04511-0].