Rapid focus map surveying for whole slide imaging with continuous sample motion.

Area of Science:

• Digital Pathology
• Microscopy Imaging

Background:

• Whole slide imaging (WSI) is cleared for primary diagnosis, but high-speed accurate focusing remains a challenge.
• Traditional WSI systems require static samples and axial scanning for focus mapping, limiting speed and efficiency.

Purpose of the Study:

• To develop a novel, high-speed focus map surveying method for WSI.
• To overcome the limitations of traditional focus mapping techniques in WSI.

Main Methods:

• Illuminating samples with two LEDs and recovering focus points using 1D autocorrelation analysis.
• Implementing a method that requires no axial scanning, additional camera, or lens.
• Enabling continuous sample motion during the focus surveying process.

Main Results:

• Demonstrated mean focusing errors of approximately 0.08 μm in static mode and 0.17 μm in continuous motion mode with a 20× objective lens.
• The method is robust for both stained and transparent samples.
• Achieved high-speed, accurate focus mapping without complex hardware.

Conclusions:

• The novel focus map surveying method offers a simple, robust, and accurate solution for WSI systems.
• This technique has the potential to standardize WSI imaging performance and benefit digital pathology.
• Applications extend to high-content microscopy, including time-lapse live-cell imaging.