Comprehensive Cell Phenotyping Method for Whole-Brain Tissue Mapping Using Highly Multiplexed Immunofluorescence Imaging

Nature Communications

Published March 10, 2021

Dragan Maric, Jahandar Jahanipour, Rebecca LI, Aditi Singh, Aryan Mobiny, Hien Van Nguyen, Andrea Sedlock, Kedar Grama & Badrinath Roysam

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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