Autofocus methods of whole slide imaging systems and the introduction of a second-generation independent dual sensor scanning method

Abstract

Accurate focusing is a critical challenge of whole slide imaging, primarily due to inherent tissue topography variability. Traditional line scanning and tile-based scanning systems are limited in their ability to acquire a high degree of focus points while still maintaining high throughput. This review examines limitations with first-generation whole slide scanning systems and explores a novel approach that employs continuous autofocus, referred to as independent dual sensor scanning. This "second-generation" concept decouples image acquisition from focusing, allowing for rapid scanning while maintaining continuous accurate focus. The technical concepts, merits, and limitations of this method are explained and compared to that of a traditional whole slide scanning system.

Keywords: Autofocus; digital pathology; whole slide imaging.

Figure 1

Tissue topography. (a) Topographical map of a typical FoV from a 5 μm thick tissue section illustrating variations in the z-plane of best focus. Multiple z-planes were acquired and composited to reconstruct the topography. (b) Graphical representation of z-dimension variations per FoV across a whole slide image. Each acquired FoV was stitched together to show the variation that occurs from tile to tile in the z-dimension. A single tile can vary over 1μm in the z-dimension from a neighboring tile. Stage tilt is easily observed (red to blue) which further contributes to variations in the z-dimension across a whole tissue section.

Figure 2

Reflective vs. image-based autofocus schemes. Reflective-based approaches set focus a fixed distance above the reference surface (glass slide surface). Image-based approaches sample images at several different z-planes and apply a figure of merit calculation to determine the optimal focal plane

Figure 3

Image-based auto-focusing approaches. A section of tissue with green cross hairs representing the focus points used to calculate a focus map. The blue dotted line (focus map) is the calculated focal plane interpolated between focus points. Red boxes are the focal plane for each field of view and each one can be adjusted in the z-position during a scan. Line scanners have more ability to adjust the z-depth during scanning. Both line and tile scanners can incorrectly predict focus between focus points. Focusing on every tile increases chances of having correct focus throughout the scan

Figure 4

Predictive auto-focusing in IDS scanning. (a) An IDS scanning system consists of a two cameras, one of which is a high speed autofocus (AF) camera and the other of which is a high resolution imaging camera. A single optical path from the sample is split into two cameras by a beam splitter. (b) The focus sensor acquires three images in different z-planes. The system calculates the optimal focus position. Once positioned there, the main imaging camera takes a high resolution image of the sample. While the main imaging camera is reading out, the process is repeated. (c) Because the system is in continuous motion, the three AF images only have a small region of overlap. The system uses the overlapping region to calculate the best focal plane