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Mapping biological processes in brain tissues requires piecing together numerous histological observations of multiple tissue samples. We present a direct method that generates readouts for a comprehensive panel of biomarkers from serial whole-brain slices, characterizing all major brain cell types, at scales ranging from subcellular compartments, individual cells, local multi-cellular niches, to whole-brain regions from each slice. We use iterative cycles of optimized 10-plex immunostaining with 10-color epifluorescence imaging to accumulate highly enriched image datasets from individual whole-brain slices, from which seamless signal-corrected mosaics are reconstructed. Specific fluorescent signals of interest are isolated computationally, rejecting autofluorescence, imaging noise, cross-channel bleed-through, and cross-labeling. Reliable large-scale cell detection and segmentation are achieved using deep neural networks. Cell phenotyping is performed by analyzing unique biomarker combinations over appropriate subcellular compartments. This approach can accelerate pre-clinical drug evaluation and system-level brain histology studies by simultaneously profiling multiple biological processes in their native anatomical context.
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