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

TL;DR

This paper introduces a rapid, continuous focus mapping method for whole slide imaging that eliminates the need for axial scanning and additional hardware, enabling high-speed, accurate focus in digital pathology.

Contribution

It presents a novel focus map surveying technique that works during continuous sample motion without axial scanning or extra equipment, applicable to brightfield and fluorescence WSI.

Findings

Achieved mean focusing error of ~0.08 microns in static mode

Achieved mean focusing error of ~0.17 microns in continuous motion mode

Compatible with stained and transparent samples, suitable for high-speed WSI

Abstract

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