Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors

Abstract

Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery

“…We correlated FD-FLIM measurements with the respective PpIX signal contribution as a ground truth. Autofluorescence lifetimes were found to be in the range of about 0.8 to 2 ns (32,33), which was substantiated by a non-pathological sample within this study. The multimodal approach revealed how even weak PpIX concentrations in tissue led to an increase of the measured lifetime respective to tissue autofluorescence.…”

“…Based on our previous results (32,35), we hypothesize that weak PpIX concentrations as found in LGG and weakly infiltrated tumor tissue of HGG increase the fluorescence lifetime respective to non-pathological brain parenchyma when measured with frequency-domain (FD)-FLIM. To investigate the extent to which PpIX fluorescence can be distinguished from a dominating autofluorescence background, we developed a multimodal surgical microscope.…”

Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors

“…These data indicate that the tumor affects the biochemical state of surrounding tissue. For the rat models, it is no surprise that healthy brain tissues in the tumor-bearing animals are affected because the size of the tumor is large, so both the infiltration of the tumor cells in the brain and the mechanical pressure of the tumor on the surrounding tissues are inevitable ( 4 , 50 , 51 ).…”

“…sodium fluorescein or indocyanine green) has proven to be a useful technique to improve the resection, but the challenges associated with non-specific distribution of the fluorophore, timing and the subjective assessment of fluorescence still remain ( 3 ). In addition, the use of 5-ALA in most cases does not allow to detect low grade gliomas, although it seems to be possible based on the fluorescence lifetime information ( 4 ). To avoid non-specific distribution of the fluorophores, fluorescent reporters targeting epidermal growth factor (EGF) receptor or other tumor specific molecules have been reported ( 5 , 6 ).…”

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

“…Autofluorescence FLIM of ex vivo tissues have also been investigated this year. Erkkila et al showed that FLIM of NAD(P)H, and protoporphyrin identify tumor margins in low-and highgrade human gliomas 31 [Fig. 3(a), right panel].…”