Spatial organization and mitigation of autofluorescence in multiplexed spatial proteomics of aged fresh-frozen human brain
- 1KTH Royal Institute of Technology - SciLifeLab
Abstract
Multiplexed imaging technologies are transforming the study of human tissue biology, but their application to the aged brain is hindered by autofluorescence, particularly in fresh-frozen specimens. Here, we characterized autofluorescence across four brain regions from 21 donors and found broad spectral emission, regional and gray-white matter differences, and an association with donor age. Photobleaching conditions adopted from formalin-fixed paraffin-embedded tissue caused marked region- and compartment-dependent damage in fresh-frozen sections. We therefore developed a Tris-EDTA-supplemented photobleaching workflow that reduced autofluorescence by 58-70% while preserving tissue architecture and cellular content. We established a custom 28-plex DNA-barcoded antibody panel targeting neuronal, glial, immune, and vascular markers, providing a resource for fresh-frozen human brain. Integration of the optimized photobleaching workflow with this panel enabled spatial proteomic analysis across fresh-frozen brain regions. By co-registering pre-photobleaching autofluorescence with multiplexed protein maps, we further established a cellular-resolution framework for spatial characterization of autofluorescence. This revealed region-dependent protein marker relationships and preferential enrichment of autofluorescent particles near nuclei and within microglial and CD68-positive regions. Together, this work establishes a practical workflow for multiplexed spatial proteomics in fresh-frozen brain and characterizes autofluorescence as both a technical confound and a spatially structured feature of the aged human brain.
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BibTeX
@misc{sutevski2026spatial,
title = {{Spatial organization and mitigation of autofluorescence in multiplexed spatial proteomics of aged fresh-frozen human brain}},
author = {I. Sutevski and L. Guo and N. Branzell and P. E. Andren and P. Svenningsson and B. Ayoglu},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.11.750056},
url = {https://www.biorxiv.org/content/10.64898/2026.09.11.750056v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Spatial organization and mitigation of autofluorescence in multiplexed spatial proteomics of aged fresh-frozen human brain
AU - I. Sutevski
AU - L. Guo
AU - N. Branzell
AU - P. E. Andren
AU - P. Svenningsson
AU - B. Ayoglu
PY - 2026
DA - 2026/09/11
PB - bioRxiv
DO - 10.64898/2026.09.11.750056
UR - https://www.biorxiv.org/content/10.64898/2026.09.11.750056v1
AB - Multiplexed imaging technologies are transforming the study of human tissue biology, but their application to the aged brain is hindered by autofluorescence, particularly in fresh-frozen specimens. Here, we characterized autofluorescence across four brain regions from 21 donors and found broad spectral emission, regional and gray-white matter differences, and an association with donor age. Photobleaching conditions adopted from formalin-fixed paraffin-embedded tissue caused marked region- and compartment-dependent damage in fresh-frozen sections. We therefore developed a Tris-EDTA-supplemented photobleaching workflow that reduced autofluorescence by 58-70% while preserving tissue architecture and cellular content. We established a custom 28-plex DNA-barcoded antibody panel targeting neuronal, glial, immune, and vascular markers, providing a resource for fresh-frozen human brain. Integration of the optimized photobleaching workflow with this panel enabled spatial proteomic analysis across fresh-frozen brain regions. By co-registering pre-photobleaching autofluorescence with multiplexed protein maps, we further established a cellular-resolution framework for spatial characterization of autofluorescence. This revealed region-dependent protein marker relationships and preferential enrichment of autofluorescent particles near nuclei and within microglial and CD68-positive regions. Together, this work establishes a practical workflow for multiplexed spatial proteomics in fresh-frozen brain and characterizes autofluorescence as both a technical confound and a spatially structured feature of the aged human brain.
ER -