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Whole slide imaging (WSI) has recently been cleared for primary diagnosis inthe US. A critical challenge of WSI is to perform accurate focusing in highspeed. Traditional systems create a focus map prior to scanning. For each focuspoint on the map, sample needs to be static in the x-y plane and axial scanningis needed to maximize the contrast. Here we report a novel focus map surveyingmethod for WSI. The reported method requires no axial scanning, no additionalcamera and lens, works for stained and transparent samples, and allowscontinuous sample motion in the surveying process. It can be used for bothbrightfield and fluorescence WSI. By using a 20X, 0.75 NA objective lens, wedemonstrate a mean focusing error of ~0.08 microns in the static mode and ~0.17microns in the continuous motion mode. The reported method may provide aturnkey solution for most existing WSI systems for its simplicity, robustness,accuracy, and high-speed. It may also standardize the imaging performance ofWSI systems for digital pathology and find other applications in high-contentmicroscopy such as DNA sequencing and time-lapse live-cell imaging.

Rapid focus map surveying for whole slide imaging with continues sample motion