Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 mu m below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.