## 3 Results  
### Optimizing the FD-FLIM Detection Band for Enhanced Delineation of PpIX from Tissue Autofluorescence  
When detecting weak PpIX concentrations in fluorescence-guided surgery, the autofluorescence background of the brain can be stronger than the actual PpIX fluorescence. This is illustrated with a representative LGG shown in Figure 1. Upon excitation with 405 nm, the fluorescence of the specimen captured with a scientific camera (>430 nm) was white to faint blue (al). Tissue autofluorescence dominated the overall signal, and the red PpIX fluorescence could not be observed visually during surgical resection and imaging. Dark red spots were seen on areas covered with blood. Spectroscopic measurements (aII) were acquired on three selected ROIs, indicated through red squares. Small peaks at 635 nm \begin{table}
\begin{tabular}{l c c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**Metastases**} & \multicolumn{3}{c}{**Low-Grade Gliomas**} & \multicolumn{3}{c}{**High-Grade Gliomas**} & \multicolumn{3}{c}{**Meningiomas**} \\ \hline
**Subgroup** & **No 5-ALA** & **Fluo-** & **Fluo+** & **No 5-ALA** & **Fluo-** & **Fluo-** & **Fluo+** & **No 5-ALA** & **Fluo-** & **Fluo+** \\ \hline filter & & & & 590 nm-740 & mm/\textgreater{}615 nm & & & & & \\ N samples & 6/6 & 5/5 & 10/10 & 4/4 & 10/10 & 19/19 & 37/37 & 20/20 & 3/3 & 16/16 \\ median r/ns & 2.5/2.5 & 4.3/4.9 & 8.4/9.8 & 2.3/2.4 & 4.7/5.4 & 2.9/3.3 & 13.4/14.1 & 2.4/2.2 & 42/3.8 & 13.2/13.8 \\ Q1 r/ns & 2.3/2.1 & 4.2/4.8 & 7.7/8.8 & 1.9/1.9 & 4.0/4.6 & 2.5/2.7 & 12.2/13.0 & 2.2/2.0 & 3.7/3.4 & 11.0/12.1 \\ Q2 r/ns & 3.0/3.3 & 4.4/5.0 & 9.7/11.3 & 3.0/3.3 & 5.5/6.2 & 3.6/4.5 & 14.1/14.6 & 3.0/2.7 & 4.5/4.7 & 14.2/14.6 \\ \(p\)-value & 0.47 & 0.03 & 0.11 & 0.44 & 0.17 & 0.18 & 0.04 & 0.9 & 0.67 & 0.15 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Fluorescence lifetime statistics for metastases, low-grade gliomas, high-grade gliomas and meningiomas, measured with a 590 nm-740 nm bandpass and a 615 nm long-pass filter. The ex vivo imaged human brain tumor specimens were further grouped into samples without 5-ALA administration (No. 5-ALA) and samples with 5-ALA administration without or with visible intraoperative fluorescence (Fluo-, Fluo+, respectively). and slightly below 700 nm were observed above the autofluorescence background, corresponding to weak accumulations of PpIX. A representative haematoxylin and eosin (H&E) stain of the sample (aIII) showed CNS tissue with increased cellularity and infiltrating pleomorphic glial tumor cells. The emission band from 590 nm to 740 nm, which we also employed in previous work, is illustrated in (bl). Signal contributions below the fitted line contributed to the autofluorescence background and were colored in blue. PpIX signal contributions were colored in red. The demodulated fluorescence intensity (mV\({}_{RMS}\)) and lifetime (ns) measured with this filter are shown in (bII) and (bIII). While the mean fluorescence intensity across the sample was 8.8 mV\({}_{RMS}\), fluorescence lifetimes averaged over ROI 1 to 3 were 2.0 ns, 4.9 ns and 3.7 ns, respectively. In the measured PpIX emission spectra, a considerable amount of autofluorescence was observed at wavelengths between 590 nm and the actual PpIX emission peak. To determine the optimized cut-on wavelength employed in this work, we derived the fluorescence emission spectrum of PpIX (data taken from PhotochemCAD [30]) and chose the wavelength with an available off-the-shelf filter closest to the point where the derivation began to rise. This resulted in setting the cut-on at 615 nm as shown in Figure 1 (cI). Cutting away autofluorescence below 615 nm decreased the average fluorescence intensity to 7.1 mV\({}_{RMS}\) (cII). This in turn increased the relative contribution of PpIX to the overall signal. As a result, the average fluorescence lifetime of ROI 1 to 3 was increased to 2.2 ns, 5.6 ns and 4.3 ns, respectively (cIII).