Single-shot autofocusing based on color-multiplexed phase detection for whole slide imaging.

Whole slide imaging (WSI) is an essential diagnostic tool in digital pathology. Conventional WSI systems acquire a z-stack of sample images along the axial direction and determine the best focal position using a focus measure function. However, this approach introduces significant overhead time and a necessity for high positional accuracy of the mechanical system. In this work, we report a novel, to the best of our knowledge, single-shot autofocusing scheme based on color-multiplexed phase detection. We place a color-multiplexed, two-pinhole-modulated camera at the epi-illumination arm. The red and green channels of the captured color image contain two copies of the sample separated by a certain distance. By maximizing the image mutual information between these two channels, we can identify this distance and subsequently recover the defocus distance of the sample without z-scanning. In addition, we replace the precise z-stage with an electrically tunable lens (ETL) at the primary imaging arm for precise axial positioning. The proposed system offers a turnkey solution that integrates real-time autofocusing capacity with an affordable mechanical system, facilitating broad accessibility and usability without compromising performance.