The loss of cellular volume during the initial phase of the PDT is attributed to water, intracellular ions and other cytoplasmic content being released by the cell membrane to maintain a normal cell density. Additionally, degraded macromolecules are expected to be discarded from the cell, indicating the early stage of apoptosis . The observed shrinking implies that hypericin-induced PDT of glioma cells initially triggers apoptosis. As the PDT progresses, the formation of large membrane blebs was observed. These are caused by a detachment of the membrane-cytoskeleton (actomyosin cortex) . Membrane blebs are morphologically characterized by a bubble-like protrusion on the cell surface (51) that can expand and retract over time . During bleb expansion, the pressure inside the cell temporarily increases and cytoplasm from the cell body flows into the bleb . This type of membrane blebbing is reported to be a typical feature of apoptotic cell death. Other bleb types can appear for necrosis, which are described to be larger, more transparent and non-retractable in comparison to apoptosis and are caused by an ion influx that causes water to enter the cell . The observation of partly small, retracting blebs, which expanded to form larger protuberances during hypericin-induced PDT would suggest that initially apoptosis is triggered, which is followed by necrosis as the PDT progresses. The assumption of an initial apoptotic and continued necrotic cell death mechanism is also supported by the observed breakdown of membrane stability and flooding of the cells with the surrounding medium (Fig. : between M6 and M7, Fig. : between M5 and M6). Widefield fluorescence images revealed that the rupture of the membrane resulting in the release of cytosolic content occurs in an explosive manner (Fig. ). The abrupt pressure release due to membrane rupture is defined to be a necrotic attribute, which further emphasizes the previously proposed mechanism of cell death . After membrane disruption, large amounts of PI diffuse into the cell at once intercalating into DNA/RNA fragments in the cytoplasm and nucleus, marking the final stage of necrotic cell death, where no apoptotic bodies are observed. As the PDT progresses, decomposition of the ER is observed until finally only its fragmented remains can be recognized. This is to be expected, as hypericin is found to accumulate in the ER, thus resulting in the ER being the main region of PDT-induced ROS production and thereby a significant level of ER stress during PDT. The ER as an important reaction site in hypericin PDT is highly linked to mitochondria via mitochondria-associated membranes (MAMs) that are stated to play a role in various cell death mechanisms in cancer, including apoptosis and necrosis . The activated cell death mechanism is supposed to be strongly dependent on the stressors, availability of certain compounds in the ER and mitochondria and on cellular, as well as environmental conditions . ER stress is reported to primarily induce apoptosis , but was in some cases also shown to cause necrosis . In conclusion, based on the analysis of FLIM composite images, spatially resolved spectroscopy and widefield fluorescence microscopy, apoptotic and necrotic features were observed for hypericin-induced PDT on glioma cells, suggesting apoptosis to be the initial mechanism followed by necrosis after sustained irradiation.