High Sensitivity and Specificity Detection of Protoporphyrin IX Fluorescence in Glioblastoma by Means of Simultaneous 2-photon Microscopy and Stimulated Raman Histology

Abstract

Abstract INTRODUCTION Extent of resection has been recognized as an important predictor for survival in glioblastoma (GBM) patients. The detection of residual infiltrating tumor at the time of surgery can be challenging. 5-aminolevulinic acid (5-ALA) has recently been approved for the visualization of malignant tissue during glioma surgery. A limitation of such optical contrast agents when imaged with standard operating microscopes is sensitivity and specificity for detecting malignant tissue with low cellularity at the infiltrative tumor margin or in low-grade gliomas. METHODS We present the use of a clinical laser microscopy system (Invenio Imaging Inc, NIO Laser Imaging System) to image fresh biopsy specimens in the OR in under 3 min based on Stimulated Raman Histology. The system was modified to simultaneously detect the 2-photon fluorescence signal of 5-ALA induced protoporphyrin IX (PpIX) fluorescence with a highly sensitive photo-multiplier tube (Hamamatsu Inc.) and a 640/70 nm emission filter (Chroma Inc.). Images were quantified with a custom cell-counter for PpIX fluorescence positive cells developed in Python. The system was validated with both 5-ALA treated and untreated invasive, human GBM xenografts. RESULTS We demonstrate for the first time the detection of PpIX positive tumor cells with single-cell sensitivity. We also demonstrate that co-use with stimulated Raman scattering to image the tissue, histology can identify auto-fluorescent background that is not associated with cells. Finally, we compared the accuracy of the automated cell-counter to manual cell-counting in a series of animals (10). CONCLUSION The combination of 2-photon fluorescence imaging of PpIX with stimulated Raman histology provides unprecedented sensitivity and specificity at the cell level. This approach may provide intraoperative real-time information for tumor detection at the infiltrative margin for maximal safe resection.