Multimodal imaging to explore endogenous fluorescence of fresh and fixed human healthy and tumor brain tissues

To complement a project toward label‐free optical biopsy and enhanced resection which the overall goal is to develop a multimodal nonlinear endomicroscope, this multimodal approach aims to enhance the accuracy in classifying brain tissue into solid tumor, infiltration and normal tissue intraoperatively. Multiple optical measurements based on one‐ and two‐photon spectral and lifetime autofluorescence, including second harmonic generation imaging, were acquired. As a prerequisite, studying the effect of the time of measurement postexcision on tissue's spectral/lifetime fluorescence properties was warranted, so spectral and lifetime fluorescences of fresh brain tissues were measured using a point‐based linear endoscope. Additionally, a comparative study on tissue's optical properties obtained by multimodal nonlinear optical imaging microscope from fresh and fixed tissue was necessary to test whether clinical validation of the nonlinear endomicroscope is feasible by extracting optical signatures from fixed tissue rather than from freshly excised samples. The former is generally chosen for convenience. Results of this study suggest that an hour is necessary postexcision to have consistent fluorescence intensities\lifetimes. The fresh (a,b,c) vs fixed (d,e,f) tissue study indicates that while all optical signals differ after fixation. The characteristic features extracted from one‐ and two‐photon excitation still discriminate normal brain (a,d) cortical tissue, glioblastoma (GBM) (b,e) and metastases (c,f).