Segmentation-before-Staining Improves Structural Fidelity in Virtual IHC-to-Multiplex IF Translation

Junhyeok Lee; Han Jang; Heeseong Eum; Joon Jang; Kyu Sung Choi

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arXiv · 2603.16160

Segmentation-before-Staining Improves Structural Fidelity in Virtual IHC-to-Multiplex IF Translation

Junhyeok Lee·Han Jang·Heeseong Eum·Joon Jang·Kyu Sung Choi

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Abstract

Multiplex immunofluorescence (mIF) enables simultaneous single-cell quantification of multiple biomarkers within intact tissue architecture, yet its high reagent cost, multi-round staining protocols, and need for specialized imaging platforms limit routine clinical adoption. Virtual staining can synthesize mIF channels from widely available brightfield immunohistochemistry (IHC), but current translators optimize pixel-level fidelity without explicitly constraining nuclear morphology. In pathology, this gap is clinically consequential: subtle distortions in nuclei count, shape, or spatial arrangement propagate directly to quantification endpoints such as the Ki67 proliferation index, where errors of a few percent can shift treatment-relevant risk categories. This work introduces a supervision-free, architecture-agnostic conditioning strategy that injects a continuous cell probability map from a pretrained nuclei segmentation foundation model as an explicit input prior, together with a variance-preserving regularization term that matches local intensity statistics to maintain cell-level heterogeneity in synthesized fluorescence channels. The soft prior retains gradient-level boundary information lost by binary thresholding, providing a richer conditioning signal without task-specific tuning. Controlled experiments across Pix2Pix with U-Net and ResNet generators, deterministic regression U-Net, and conditional diffusion on two independent datasets demonstrate consistent improvements in nuclei count fidelity and perceptual quality, as the sole modifications. Code will be made publicly available upon acceptance.

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Junhyeok Lee, Han Jang, Heeseong Eum, Joon Jang, Kyu Sung Choi. 2026-03-17. Segmentation-before-Staining Improves Structural Fidelity in Virtual IHC-to-Multiplex IF Translation. https://arxiv.org/abs/2603.16160

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