Metric-driven Pre-acquisition Diagnostics for Automated Microscopy Ultraviolet Surface Excitation (MUSE)

Abstract

Background Automated microscopy enables large-scale tiled imaging but remains sensitive to variations in alignment, illumination, focus, and sample coverage that may only be detected after acquisition. We present an adaptive pre-acquisition quality-control framework for microscopy ultraviolet surface excitation (MUSE) that uses sparse scout imaging and tile-level metrics to guide acquisition and selective rescanning. Methods The framework quantifies brightness, dynamic range, focus, tissue fraction, and exposure error across the field of view. For example, in a representative 10 x 10 acquisition (100 tiles, 20-megapixel imagery), the mean p99 brightness was 0.525 (CV 10.4%), the mean dynamic range was 0.331, the median focus score was 0.0333, and the median tissue fraction was 0.943. Furthermore, applying a conservative exposure-error threshold (|error| > 0.25) identified 25% of tiles as rescan candidates. Results These results show that compact tile-level metrics can be converted into actionable quality gates, enabling early detection of local acquisition errors and targeted rescanning to improve mosaic consistency while minimizing unnecessary acquisition.