Two-photon volumetric study of cleared, invasive ductal carcinoma breast tissue samples and associated axillary lymph nodes

BackgroundBreast cancer remains the most frequently diagnosed cancer among women worldwide, with invasive ductal carcinoma (IDC) representing the most common subtype. Despite its high prevalence, current diagnostic workflows rely on 5 {micro}m-thin haematoxylin and eosin-stained (H&E) sections, inherently limiting spatial insight into tumor architecture and extracellular matrix (ECM) organization. As the interest in studying intratumoral heterogeneity increases, three-dimensional (3D) imaging is becoming an increasingly valuable tool. MethodsThis study applied a tissue clearing and imaging pipeline to large formalin-fixed paraffin-embedded (FFPE) breast and lymph node tissue using IDC samples from Maastricht University Medical Centre+. Deparaffinized samples were processed using a modified MASH protocol. Tissues were labelled with Neutral Red, Eosin Y, Methyl Green, and DAPI. Tissue shrinkage was analysed across all processed samples. Two-photon (2P) microscopy was used to image malignant and non-malignant breast tissue, and matched axillary lymph nodes from a patient with grade I IDC to depths of up to 1000 {micro}m via DAPI and second harmonic generation (SHG) channels. Image analysis included assessments of dye penetration, nuclear and collagen content, and fiber orientation using FIJI software. ResultsClearing and staining preserved tissue structure and achieved high transparency across millimetre-scale volumes. 2D surface shrinkage averaged 6.7% (p < 0.001). DAPI signal penetration was consistent with SHG signal profiles up to approximately 600 {micro}m of tissue depth. Structures such as terminal ductal lobular units, adipocytes, vasculature, and lymphoid follicles were clearly visualized in 3D. Quantitative and qualitative analysis in grade I IDC tissue revealed regional differences in cell size and shape, collagen content, and fiber coherency, indicating localized early-stage ECM remodelling. ConclusionThis is the first study to apply 2P microscopy with MASH clearing to large FFPE IDC breast and lymph node samples. The protocol enables reproducible high-resolution volumetric imaging and lays the groundwork for future research applications, leading to potential diagnostic applications.