## 4 Discussion
Note also that the reduced overall signal intensity in the optimized filter (17% less compared to the previous filter) may contribute to an increase of phase noise and thereby a reduction of the signal-to-noise ratio for tissue exhibiting very weak fluorescence, e.g., parts of the specimens covered with blood. The overall photon budget, however, is mainly determined by the detection efficiency of the system and could be tailored to an acceptable level of phase noise. The influence of the integrated signal on noise in FD-FLIM phase measurements is described in more detail in the supplementary materials of our previous work [15]. We furthermore emphasize that of the 37 specimens throughout all tumor entities lacking visible fluorescence during surgery, all specimens could be distinguished from the 1 ns to 2 ns expected for non-pathological brain by increased fluorescence lifetimes. Note that imaging of specimens to compare filters had to be performed consecutively, which might have led to bleaching of fluorescence during the first measurement and affected the second acquisition. We analyzed bleaching by consecutively imaging a specimen with the same filter and found that bleaching-induced lifetime reduction between two scans was smaller than 0.1 ns. Additionally, we varied the order of which filter was used to image first between specimens and could not find any influence on the imaging order. The overall coherence of what was observed in all groups and the multimodal FD-FLIM and spectroscopy evaluation provided evidence to favor the 615 nm long-pass filter. Potential further optimization of the PpIX to autofluorescence ratio would be to restrict the detection band to the main PpIX emission peak. We performed a first test with a 620 nm to 640 nm bandpass filter, which slightly increased the measured lifetime with respect to the 615 nm long-pass filter at the cost of a reduced signal to noise ratio and increased phase noise in our measurements. Additionally, a two-peaked emission of PpIX was observed in LGG, with one physicochemical state peaking at a lower wavelength of 620 nm [38]. Hence, a cut-on wavelength at 615 nm was the preferred choice. While we employed an off-the-shelf long-pass filter, a bandpass filter with a cut-off at around 715 nm, which is right after the second PpIX emission peak, seems to be the optimal choice for FD-FLIM imaging of PpIX. Spectrally resolved FLIM would be highly beneficial to investigate this in more detail [39]. A valuable additional frequency-domain metric in future studies would be the modulation lifetime \(\tau_{m}\)[28]. Thus, a measure of the ground-state heterogeneity could be given by \((\tau_{m}/\tau_{\phi})\) -1, essentially reflecting the lifetime variance of the multi-exponential fluorophore decays within the detected spectrum. For higher-grade tumors, where PpIX fluorescence outweighs tissue autofluorescence, this measure would be expected to approach 0 as \(\tau_{\phi}=\tau_{m}\) for mono-exponential decays [28]. For lower-grade tumors, where PpIX is on the order of magnitude of tissue autofluorescence, the ground state heterogeneity could be an interesting additional metric for distinguishing 5-ALA labeled tumors from non-pathological brain. Additionally, a lifetime based ratio of PpIX to background fluorescence could be given, e.g., by transforming phase and modulation lifetime to the vector space of phasor plots [24; 40]. Furthermore, time-domain FLIM would allow for better contrasting PpIX from the autofluorescence background by fitting a multi-exponential model. Fluorescence anisotropy measurements of rotational correlation time potentially would provide an additional source of contrast for resolving ground state heterogeneity in 5-ALA labeled tumors and non-pathological brain. Yet, as outlined in this and our previous works [15; 22; 23; 24; 25], the average phase lifetime in FD-FLIM alone does constitute a sensitive and promising approach for PpIX fluorescence guidance.