Tertiary lymphoid structures (TLS) are immune aggregates that play a central role in cancer prognosis and response to immunotherapy. Yet, in traditional 2D pathology, their true frequency and complexity often go undetected.

With Alpenglow’s 3Di platform and computational H&E staining (TO-PRO-3 nuclear stain + eosin), TLSs in NSCLC samples can be visualized and quantified in three dimensions without sectioning.

The Scout-to-Zoom workflow makes this possible:

• Scout mode rapidly scans whole blocks at 2 µm resolution to identify TLS and other features across the tissue landscape.

• Zoom mode then images regions of interest at subcellular resolution (0.167 µm), enabling single-cell segmentation and precise TLS characterization.

In one NSCLC biopsy, Scout imaging revealed dense nuclei clusters that, when examined in Zoom mode, were confirmed as two distinct TLSs buried 200 µm below the surface. Their unique volumes and surface areas were quantified, data that would have been invisible in conventional 2D histology.

Why it matters:

• Accurate TLS detection: avoid miscounts caused by thin cross-sections

• Quantitative insights: measure TLS volume, surface area, and cellular density in 3D

• Biological context: analyze TLS maturity, proximity to vasculature, and relationships to tumor and stroma

• Translational impact: TLS density and maturity are emerging biomarkers of immunotherapy response across cancers

By combining computational H&E staining with 3D tissue imaging, researchers gain a deeper understanding of tumor-immune interactions, which are critical for biomarker discovery and the development of next-generation cancer therapies.

Explore the full case study: 3D Computational H&E Imaging of TLS in NSCLC