Nature丨Multinational Team Develops Comprehensive Disease Map Achieving 95 Rounds of Trillion-Pixel Data Analysis, Revealing Kidney Disease Protein Distribution and Mechanisms}
A global team created an advanced disease map using 95 rounds of trillion-pixel imaging, uncovering kidney protein distributions and disease mechanisms with unprecedented high-resolution data analysis.
Imagine compressing an entire city’s nightscape into a postage stamp, yet only illuminating four streetlights—that’s the challenge of traditional immunofluorescence: at most 3-4 proteins, like painting Beijing with four watercolor brushes.
This limitation stems from antibody combination complexity and image resolution constraints, which restrict the scope of image analysis. Recently, researchers from Aarhus University in Denmark and the Center for Molecular Neurobiology Hamburg (ZMNH) in Germany introduced PathoPlex, a pathology-oriented multiplexing framework that not only solves these issues but also reveals the spatial distribution of proteins within tissues in an unprecedented way.
The study titled "Pathology-oriented multiplexing enables integrative disease mapping" was published on Nature on July 18, 2025.
Figure: PathoPlex (Source: the paper).
The core advantage of PathoPlex lies in its combination of high-throughput and high-resolution imaging, integrating immunofluorescence staining with image analysis to detect multiple proteins’ spatial distribution at subcellular resolution.
In one experiment, researchers performed up to 95 rounds of imaging, mapping over 140 proteins at a pixel resolution of 80 nanometers. Over the course of the experiment, they generated more than 600 billion pixels of data, revealing multi-layered, multi-dimensional protein expression patterns—an achievement that is still not at its limit.
Illustration: PathoPlex (Source: the paper).
To efficiently handle such massive data, the team developed spatiomic, an open-source software package that leverages GPU acceleration for image registration, dimensionality reduction, and clustering, making data processing no longer a bottleneck.
Validation and Concept Proof
Quality control standards for PathoPlex were first established in mouse tissues and then extended to human samples.
The first level of control involves continuous antibody panel imaging to prevent incomplete elution, which could cause cross-reactivity or residual signals in subsequent cycles.
The second level involves direct imaging after elution to confirm the absence of fluorescence signals. The third includes imaging with secondary antibodies without primary antibody incubation to ensure no residual activity, adding an extra layer for image analysis.
The fourth level involves successful re-staining after multiple cycles, confirming epitope preservation and effective antibody elution. This strategy achieved near-perfect elution rates within just two cycles, with effective re-staining after 60 cycles.
After all imaging cycles, images are aligned to compensate for any shifts across cycles.
Next, the team used PathoPlex to analyze the progression of acute injury to crescentic glomerulonephritis (CGN) in mice.
Illustration: Identification of epithelial JUN activity as a key switch in immune-mediated kidney disease (Source: the paper).
In the experiment, 34 markers were used to acquire approximately 5 billion pixels at 80 nm resolution. Additional histological slices from the same animals were evaluated blindly by two experienced nephropathologists, with results consistent with the framework.
For diabetic kidney disease (DKD), PathoPlex revealed calcium-related stress phenomena in renal tubules, which may be early markers of kidney damage in diabetes.
Comparing diabetic and normal kidney tissues, researchers found that stress markers like calcium channel proteins were significantly upregulated in diabetic kidneys, even before structural changes appeared, indicating early molecular alterations.
Illustration: PathoPlex as a tool for analyzing human DKD (Source: the paper).
These findings support the use of PathoPlex for early biomarker validation and intervention strategies in diabetic nephropathy.
Broader Precision Medicine Applications
The advent of PathoPlex not only provides a new research tool but also lays a solid foundation for precision medicine. Its high-throughput imaging and powerful data analysis enable in-depth exploration of protein distribution, expression patterns, and interactions within tissues.
However, challenges remain, such as the high costs of each imaging cycle and data processing, as well as maintaining data consistency and quality as antibody panels expand.
Future research should focus on automating image analysis to handle larger, more complex datasets. With ongoing technological improvements and expanding applications, PathoPlex is expected to become a vital tool in precision medicine, supporting early diagnosis, treatment, and drug development.