Rebuilding the Microscope for Digital Pathology

Visual examination of biopsy sections by microscopy is the gold standard for cancer diagnosis. But with the improvements in digital imaging over the past decade, there has been a worldwide upsurge in attention on digital pathology – which promises to make the prediction, diagnosis, and prognosis of cancers and other diseases better, faster and cheaper than ever. In particular, digital pathology has become a popular alternative for secondary consultation with a remote specialist due to the time saved by sharing digital images instead of transferring glass slides. That time saving has turned digital pathology from a “blue-sky” approach into a promising field of diagnostic medicine. And it will only continue to grow with the advent of a new generation of pathologists trained on digital images and the emergence of artificial intelligence in medical diagnosis.

Digital pathology currently employs whole slide imaging (WSI) systems with high-resolution objective lenses to digitize histology sections. These WSI systems use high-speed mechanical scanning to generate gigapixel images of entire histology slides. The resulting images are complete enough to provide a quick overview of an entire section, but detailed enough to provide close-up views of areas of interest and accommodate automatic image analysis. But despite these advantages, WSI systems face several challenges. For instance, their high-magnification lenses provide the resolution required to resolve structural details, but their shallow depth of field makes acquiring in-focus images of sections with uneven topography difficult. To overcome this shortfall and reduce the need to re-scan slides, WSI systems perform focus map surveying or Z-stack imaging. Unfortunately, neither of these approaches is ideal, as focus maps can only reduce (not eliminate) out-of-focus areas, and Z-stack images create large files that are hard to view, share or archive. Finally, the need for precise mechanical movements and feedback control necessitate expensive hardware – current WSI systems can cost as much as US$150,000! The cost of acquiring and maintaining such a system is a significant barrier to adoption by hospitals, clinics, and pathology groups.

Recently, a novel microscopy technique, Fourier ptychography (1)(2)(3), has been developed to acquire high-resolution, wide field-of-view images without mechanical scanning. This technique uses an LED array for sample illumination and a low-magnification lens (typically a 2X objective) for image acquisition. Each LED element on the array illuminates the sample with one incident angle; for each incident angle, the device records one low-resolution intensity image. The images are then stitched together in the Fourier domain to produce a single high-resolution picture.

Unlike conventional microscopy platforms, the final achievable resolution of Fourier ptychography does not depend on the choice of objective lens; instead, it is determined by the largest incident angle of the LED array. It has been shown that this approach can use a 2X, 0.08 numerical aperture (NA) objective lens to produce an image with 0.5 synthetic NA. What does that mean? Essentially, that it combines the field-of-view of a 2X lens with the resolution of a 20X lens. The low-magnification lens also offers an additional advantage: a broad depth-of-field that can be extended even further with reconstruction algorithms – up to 0.3 mm, at least 50 times longer than that of a conventional platform with a similar numerical aperture. A simple, robust solution to WSI’s focusing problem!

Fourier ptychography is currently in its infancy, but I anticipate that it will continue to grow and expand. I look forward to seeing the new insights it brings to the development of digital pathology platforms in the future.