Further experiments were carried out, where PI was supplemented to the cultivation to act as a fluorescence marker sensitive to cell stress and death in the lifetime imaging measurements. The investigation of hypericin PDT on a single glioma cell with resorufin and PI present in the culture medium is displayed in a series of FLIM composite images in Fig. . Again, resorufin is mainly located in the surrounding medium, as demonstrated by average FLTs of 3 ns around the glioma cell. Immediately after PDT induction (Fig. ), the cell morphology is unchanged, and the glioma cell has an elongated, narrow shape. Hypericin is accumulated throughout the entire cell with the largest concentration inside the ER, as illustrated by the bright green color for hypericin FLTs of approximately 7 ns. Only miniscule amounts of PI are found inside the cell, as the selective permeability of the membrane is still intact. The contribution of the individual dyes to the overall fluorescence decay of the intact cell, and their evolution as the PDT progresses are depicted and discussed in section 6 of the SI. In the early stages of PDT, contraction of the cellular body accompanied by membrane bleb formation is observed (Fig. ). At this early stage of cellular damage, small amounts of PI are found to already enter the cell and bind to DNA in the nucleoli in the nucleus. As a result, from Fig. M2-4 the nucleoli become increasingly fluorescent with a long average FLT of 12-13 ns (red). The small surface blebs on the bottom of the cell in Fig. M3 merge and form a large protuberance in M4/M5. This is accompanied by a strong increase in PI fluorescence, as can be seen in Fig. and the FLIM image itself. The final steps of PDT (Fig. After the recording of each FLIM image presented in Fig. , fluorescence spectra were acquired at different spatial positions corresponding to certain cellular features; namely ER, nucleus, bubble and outside. The exact positions are indicated in Fig. . Each subplot of Fig. shows a 2D surface representation (left), the individual spectra (upper right) and the spectral composition (lower right) determined by spectral unmixing. In this context, the spectral composition refers to the relative contribution of the individual components to the sum spectrum and is not a direct measure of the concentrations of the dyes (see SI section 8 for more information). The spectra acquired in the ER are shown in Fig. . At time point M1, the fluorescence spectrum shows a strong signal of hypericin at 600 nm with a small contribution of resorufin (shoulder at 584 nm). In the first steps of the PDT (M1-4), the hypericin fluorescence intensity in the ER decreases, which can be attributed to a combination of direct photobleaching of hypericin due to the irradiation and bleaching due to the formation of ROS created in the immediate surrounding of the hypericin during the PDT. The triplet transition and the quenching of the triplet absorption in the presence of oxygen was confirmed with nanosecond transient absorption measurements of hypericin in DMSO and in aqueous solution which are displayed and discussed in section 9 of the SI. The fluorescence intensity of resorufin and PI are hardly altered since the selective permeability of the still intact cell membrane prevents them from entering the cell. At later stages, the contributions of resorufin and PI to the spectra significantly increase, indicating that the surrounding medium is entering the cell (increase of resorufin PL intensity) and the increasing cellular damage (increase of PI PL intensity). In Fig. , the corresponding spectra acquired in the cell's nucleus are shown. For the early measurements (M1-M4), the fluorescence intensity in this region is only approx.