Three stained sections from each sample were imaged at 10x magnification within 36 hours of staining (except for sample C, which had unacceptable artefact in one section in round 1). Whole-section multichannel images were captured using a Nikon Ti2-E motorized microscope with a Lumencor SOLA LED light engine (at 50% power), a Hamamatsu ORCA Fusion camera (in 16-bit ultra-quiet mode), and Nikon Plan Apochromat Lambda objectives. Final image resolution was 0.64 μm/pixel.
All exposures were chosen by the trained operators based on best microscopy practices of avoiding signal saturation while maximizing the useful range of intensity values detected by the camera in healthy control samples. Identical imaging settings were used for all sections within the same round of staining and imaging. Operators selected exposures independently in rounds 1 and 2, which approximates the degree of variation that is likely to arise between images collected in different laboratories using different microscopy equipment. For round 3, the imaging operator was required to use the same exposure settings as those used in round 1.
All exposures were chosen by the trained operators based on best microscopy practices of avoiding signal saturation while maximizing the useful range of intensity values detected by the camera in healthy control samples. Identical imaging settings were used for all sections within the same round of staining and imaging. Operators selected exposures independently in rounds 1 and 2, which approximates the degree of variation that is likely to arise between images collected in different laboratories using different microscopy equipment. For round 3, the imaging operator was required to use the same exposure settings as those used in round 1.