  Background: The use of whole slide imaging (WSI) for digital pathology has
recently been cleared for primary diagnosis in the US. 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.
