Rapid and robust whole slide imaging based on LED-array illumination and color-multiplexed single-shot autofocusing.

Background Digital pathology is experiencing an exponential period of growth catalyzed by advancements in imaging hardware and progresses in machine learning. The use of whole slide imaging (WSI) for digital pathology has recently been cleared for primary diagnosis in the US. The demand for using frozen section procedure for rapid identification of cancerous tissue during surgery is another driving force for the development of WSI. A conventional WSI system scans the tissue slide to different positions and acquires the digital images. In a typical implementation, a focus map is created prior to the scanning process, leading to significant overhead time and a necessity for high positional accuracy of the mechanical system. The resulting cost of WSI system is often prohibitive for frozen section procedure during surgery. Methods We report a novel WSI scheme based on a programmable LED array for sample illumination. In between two regular brightfield image acquisitions, we acquire one additional image by turning on a red and a green LED for color multiplexed illumination. We then identify the translational shift of the red- and green-channel images by maximizing the image mutual information or cross-correlation. The resulting translational shift is used for dynamic focus correction in the scanning process. Since we track the differential focus during adjacent acquisitions, there is no positional repeatability requirement in our scheme. Results We demonstrate a prototype WSI platform with a mean focusing error of ~0.3 microns. Different from previous implementations, this prototype platform requires no focus map surveying, no secondary camera or additional optics, and allows for continuous sample motion in the focus tracking process. Conclusions A programmable LED array can be used for color-multiplexed single-shot autofocusing in WSI. The reported scheme may enable the development of cost-effective WSI platforms without positional repeatability requirement. It may also provide a turnkey solution for other high-content microscopy applications.