Nanoscale-Sensitive VSFG Microscopy for Identifying Breast Tumor in FFPE Samples

Nanoscale collagen remodeling is a critical physical signature of tumor development and metastasis. Recently, we have shown that vibrational sum-frequency generation (VSFG) microscopy can detect tumor tissues prepared from Optimal Cutting Temperature (OCT) samples, where this unique sensitivity to nanostructure comes from mode-specific coherent interference. Yet, most clinical tissues are processed as formalin-fixed, paraffin-embedded (FFPE) blocks, which remains unknown whether these harsh fixations and embedding processes destroy the subtle structural cues that VSFG detects. Thus, it could present a large barrier for further application of VSFG broadly into biophysics and biomedical research of tumors. Here, we demonstrate that VSFG remains fully effective in deparaffinized FFPE tissues. Crucially, following deparaffinization, key diagnostic metrics were statistically indistinguishable from OCT cryosection controls. These metrics included VSFG spectra, chemical images, and the collagen ratio I _NHs /I _{CH2 ,Ss} . Complementary atomic force microscopy (AFM) nanomechanics corroborated this finding. These findings open the path of using VSFG imaging for standard clinical workflows and the vast global archives of FFPE tissues for retrospective prognostic studies and label-free diagnostics.