Petrographic Microscopy with Ray Tracing and Segmentation from Multi-Angle Polarisation Whole-Slide Images
Authors: Acevedo Zamora Marco Andres , Kamber Balz Samuel 
Published: January 2023

‘Slide scanners’ are rapid optical microscopes equipped with automated and accurate x-y travel stages with virtual z-motion that cannot be rotated. In biomedical microscopic imaging, they are widely deployed to generate whole-slide images (WSI) of tissue samples in various modes of illumination. The availability of WSI has motivated the development of instrument-agnostic advanced image analysis software, helping drug development, pathology, and many other areas of research. Slide scanners are now being modified to enable polarised petrographic microscopy by simulating stage rotation with the acquisition of multiple rotation angles of the polariser–analyser pair for observing randomly oriented anisotropic materials. Here we report on the calibration strategy of one repurposed slide scanner and describe a pilot image analysis pipeline designed to introduce the wider audience to the complexity of performing computer-assisted feature recognition on mineral groups. The repurposed biological scanner produces transmitted light plane- and cross-polarised (TL-PPL and XPL) and unpolarised reflected light (RL) WSI from polished thin sections or slim epoxy mounts at various magnifications, yielding pixel dimensions from ca. 2.7 × 2.7 to 0.14 × 0.14 µm. A data tree of 14 WSI is regularly obtained, containing two RL and six of each PPL and XPL WSI (at 18° rotation increments). This pyramidal image stack is stitched and built into a local server database simultaneously with acquisition. The pyramids (multi-resolution ‘cubes’) can be viewed with freeware locally deployed for teaching petrography and collaborative research. The main progress reported here concerns image analysis with a pilot open-source software pipeline enabling semantic segmentation on petrographic imagery. For this purpose, all WSI are post-processed and aligned to a ‘fixed’ reflective surface (RL), and the PPL and XPL stacks are then summarised in one image, each with ray tracing that describes visible light reflection, absorption, and O- and E-wave interference phenomena. The maximum red-green-blue values were found to best overcome the limitation of refractive index anisotropy for segmentation based on pixel-neighbouring feature maps. This strongly reduces the variation in dichroism in PPL and interference colour in XPL. The synthetic ray trace WSI is then combined with one RL to estimate modal mineralogy with multi-scale algorithms originally designed for object-based cell segmentation in pathological tissues. This requires generating a small number of polygonal expert annotations that inform a training dataset, enabling on-the-fly machine learning classification into mineral classes. The accuracy of the approach was tested by comparison with modal mineralogy obtained by energy-dispersive spectroscopy scanning electron microscopy (SEM-EDX) for a suite of rocks of simple mineralogy (granulites and peridotite). The strengths and limitations of the pixel-based classification approach are described, and phenomena from sample preparation imperfections to semantic segmentation artefacts around fine-grained minerals and/or of indiscriminate optical properties are discussed. Finally, we provide an outlook on image analysis strategies that will improve the status quo by using the first-pass mineralogy identification from optical WSI to generate a location grid to obtain targeted chemical data (e.g., by SEM-EDX) and by considering the rock texture.