## 4 Discussion  
In this work, we improved FD-FLIM PpIX visualization for fluorescence-guided surgery by minimizing autofluorescence crosstalk within the detection band. Optical filters have to be tailored to their respective application to ensure optimal performance in fluorescence imaging. This is of importance, especially when several fluorophores are excited and emitting within the detection band. In metabolic imaging of the intrinsic fluorophores nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), substantial work has been performed to optimize the filter specifications and minimize crosstalk [31, 32]. FLIM of PpIX has emerged only recently, and previous work has employed a broad detection band (590-740 nm) to capture the full PpIX emission spectrum and to increase the overall collected signal [15, 22, 23, 24, 25]. This did not fully consider the influence of tissue autofluorescence, which can be of the same order of magnitude as PpIX fluorescence for lower-grade tumors and weakly tumor-infiltrated brains. In FD-FLIM, the measured lifetime is a weighted average of all emitting fluorophores. When exciting fluorescence in the blue range of the spectrum, tissue autofluorescence of non-pathological brain above 590 nm decays in about 1-2 ns [15, 24, 25, 27], with individual contributions of flavins, lipofuscin, vitamin A, endogenous porphyrins and further fluorophores [33]. Autofluorescence lifetimes in tumorous tissue were found to be increased beyond 2 ns [34]. PpIX yields higher lifetimes with up to 16.4 ns in organic solvent [26] and about 6.3 ns [26] to 7.4 ns [35] in mitochondria. Photopotoporphyrin and the photoproducts II+III decay within 5.4 ns in mitochondria [36] and about 2-4 ns in the cytoplasm [37], respectively. Weak concentrations of PpIX in tissue can be detected by increased lifetimes with respect to the lifetimes of the autofluorescence background. A higher relative contribution of PpIX to the overall excited state population thereby increases the average lifetime of all fluorophores contributing to the multi-exponential decay [15]. Combining FD-FLIM with optical spectroscopy revealed a stronger increase of the measured lifetime starting at a RSC\({}_{PpIX}\) of about 0.25 when detecting fluorescence >615 nm. Blocking autofluorescence between 590 nm and 615 nm increased the contribution of PpIX to the overall signal, which favored distinguishing PpIX from the autofluorescence background. Similarly, descriptive statistics coherently showed increased lifetimes for all subgroups with 5-ALA administration, besides the MNG group lacking visual fluorescence during resection. For this group, we observed increased autofluorescence signal contributions in our spectral measurements. A potential explanation could be the accumulation of flavins with relatively long lifetimes, such as flavin mononucleotide (4.8 ns when bound to proteins). Removing these contributions with the 615 nm longpass filter would explain the contrary effect on the overall lifetime for MNG with low PpIX concentrations. Further studies investigating on the autofluorescence properties, especially of MNG, are warranted. Differences between the two spectral filters were not found to be significant for most of the groups, which can be contributed to a limited number of samples per group and a relatively high variance within the groups compared to the lifetime differences between the groups. Although 130 specimens were included in the study, the number of tumor entities and subgroups (visible-, not visible fluorescence, and no 5-ALA administration) further reduced the group size. Additionally, specimens from core tumor areas, infiltration zones, and necrotic and reactive areas were combined within the subgroups, which contributed to the variance within groups. Yet, the trend of increased fluorescence lifetimes for the optimized filter was very coherent for all subgroups, with the exemption of MNG group discussed above.