## 3 Results
### Optimizing the FD-FLIM Detection Band for Enhanced Delineation of PpIX from Tissue Autofluorescence
On average, the signal intensity for detection >615 nm was decreased by about 17 % for all specimens when compared to the band from 590 nm to 740 nm. Figure 1: (**a**) Fluorescence >430 nm of the specimen captured with a scientific camera (I), spectra measured on specific regions of interest on the specimen (II), and representative histopathological H&E stained scan (III). Data were acquired with an emission filter from 590 to 740 nm (b(I)) and >615 nm (c(I)). The respective demodulated root mean square fluorescence intensity (mV\({}_{RMS}\)) and lifetime (ns) maps are shown in (b(II),c(II)) and (b(III),c(III)). Minimizing Autofluorescence Crosstalk Improves the Detection of Protoporphyrin IX with Frequency-Domain FLIM  
Based on these observations, we conducted a study to investigate the effect of the adjusted filter on a larger number of specimens from various tumor entities. We acquired FD-FLIM measurements on 130 brain tumor specimens ex vivo and combined them with additional spectroscopic measurements. Spectra were used to determine the relative PpIX signal contribution (RSC\({}_{PpIX}\)) [15], which is why specimens without 5-ALA administration were excluded from the analysis in Figure 2a. Plotting the measured lifetime as a function of the RSC\({}_{PpIX}\) illustrated how blocking autofluorescence below 615 nm impacted the measured lifetime (Figure 2a). Data acquired with the 615 nm long-pass filter were drawn in yellow. Data points were labelled according  
Figure 2: Comparison of FD-FLIM PpIX detection for the spectral bands 590 nm to 740 nm and >615 nm. (**a**) Correlating the fluorescence lifetime with the relative PpIX signal contribution illustrates an increase in the measured lifetime for detection >615 nm starting around 0.25 relative PpIX signal contribution. 95% prediction intervals (dashed lines) and 95% confidence intervals (color shade around the 5 th order polynomial fit) are given both for detection from 590–740 nm and >615 nm. Fluorescence lifetimes were then compared for the two bands for metastases (**b**), low-and high-grade gliomas (**c**) and meningiomas (**d**). Samples were grouped according to the perceived fluorescence during surgery (visible, not visible), and samples without 5-ALA administration prior to surgery were treated as a separate group. Data from non-pathological specimens from our previous work [15] are indicated with gray lines (solid: median, dashed: first and third quartiles) for direct comparison to the lifetimes measured in tumorous tissue. The number of specimens per filter and subgroup is indicated at the lower end of (**b–d**) (e.g., 6/6). to their fluorescence status during surgery, with a circle indicating lack of visibility and a plus visible fluorescence. Below 0.25 RSC\({}_{PplX}\), autofluorescence dominated the overall signal with close to no alterations of the measured fluorescence lifetime between both filters. Above 0.25 RSC\({}_{PplX}\), the measured lifetime began to increase due to the increased PpIX content in the signal. Comparing the two filters, we observed a stronger increase in the measured lifetime for the cut-on at 615 nm, gradually increasing from 0.25 RSC\({}_{PplX}\) to about 0.8 RSC\({}_{PplX}\). With PpIX fluorescence and tissue autofluorescence being on the same order of magnitude, this filter impacted the fluorophore ratio in favor of PpIX. Above 0.8 RSC\({}_{PplX}\), PpIX fluorescence gradually started to become visible during surgical resection, with PpIX now being the dominant signal contributor. As a result, tissue autofluorescence had less influence on the overall signal and the differences of the measured lifetimes between both filters began to decrease. Samples with strong intraoperative fluorescence were condensed at a RSC\({}_{PplX}\) of 1.0. We then grouped all specimens in MET (Figure 2b), LGG, HGG (Figure 2c) and MNG (Figure 2d).