Protoporphyrin IX (PpIX) fluorescence-guided surgery has established as a standard for resecting malign glioma. However, low-grade glioma or sparsely infiltrated brain often emit weak PpIX fluorescence and are hard to distinguish from non-pathological tissue. Furthermore, spectrally overlapping autofluorescence inherently limits the sensitivity of fluorescence-intensity based PpIX detection. We therefore integrated frequency-domain fluorescence lifetime imaging together with a spectrometer in a surgical microscope. When analyzing human glioma samples ex vivo, weak PpIX fluorescence could be differentiated from the autofluorescence background through increased lifetimes. Characteristic peaks in the spectral measurements (635, 705nm) confirmed low concentrations of PpIX in the tissue.
Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.
Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.
Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.
Results: Our imaging system enabled macroscopic FLIM of a 6.5 × 6.5 mm2 field of view at spatial resolutions <20 μm. A frame of 512 × 512 pixels with a lifetime accuracy <1 ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology.
Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.
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