Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. Here we describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from non-neoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and H&E microscopy for detection of glioma infiltration (κ=0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Brain tissue biopsies are required to histologically diagnose brain tumors, but current approaches are limited by tissue characterization at the time of surgery. Emerging technologies such as mass spectrometry imaging can enable a rapid direct analysis of cancerous tissue based on molecular composition. Here we illustrate how gliomas can be rapidly classified by desorption electrospray mass spectrometry (DESI-MS) imaging, multivariate statistical analysis, and machine learning. DESI-MS imaging was performed on thirty-six human glioma samples, including oligodendroglioma, astrocytoma and oligoastrocytoma, all of different histologic grades and varied tumor cell concentration. Grey and white matter from glial tumors were readily discriminated and detailed diagnostic information could be provided. Classifiers for subtype, grade and concentration features generated with lipidomic data showed high recognition capability with >97% cross-validation. Specimen classification in an independent validation set agreed with expert histopathology diagnosis for 81% of tested features. Together, our findings offer proof of concept that intra-operative examination and classification of brain tissue by mass spectrometry can provide surgeons, pathologists, and oncologists with critical and previously unavailable information to rapidly guide surgical resections that can improve management of patients with malignant brain tumors.