## 4 Discussion
### Two-Photon Imaging  
Novel advances in unraveling the pathomechanisms associated with epilepsy described in this review have benefitted greatly from the advent and development of 2 PI as an imaging technique. This subsection describes specific features of 2 PI and how these features were utilized within studies included in this review. 2 PI enables high-resolution deep tissue imaging of vascular structures, neuronal cells, and fluorophores, with limited background signal [1], and is suitable for molecular imaging of living tissues or in live animals. This feature was fully taken advantage of in some of the studies included in the present review. For example, three studies [57; 59; 60] were able to visualize subtle changes in the microvasculature (namely vasodilation and vasoconstriction) that accompany epileptiform activity. In vivo 2 PI can be used to quantify hemodynamics and vascular responses after epileptiform activity. Additionally, 2 PI was even employed to measure capillary flow during epileptiform activity [60], and visualization of neurons, microglia, astrocytes, and neurites revealed morphological changes accompanying seizures. As a result of these specific features, 2 PI allowed for the identification of functional and structural alterations in epilepsy, which have not been described previously. In these in vivo instances, using confocal laser scanning microscopy would have likely been inadequate, as comparatively, 2 PI significantly reduces photobleaching, increases cell viability, and allows for deeper tissue penetration [69]. Furthermore, it has been reported that, when imaging neuronal cells specifically, single-photon microscopy suffers from increased interference by neuropil and decreased signal-to-noise ratio compared to 2 PI [70]. 2 PI is particularly well-suited for uncaging experiments, which involves the use of light to artificially activate biological compounds, such as glutamate or calcium, and therefore to increase their concentrations in small focal volumes [71]. Due to the inherent nonlinearity of the two-photon system, 2 PI is more suitable for uncaging than conventional single-photon excitation [72]. Two-photon uncaging produces excited states exactly as UV excitation does while also overcoming major limitations when probing biological tissue, such as spatial resolution, tissue penetration, and toxicity [73]. Two-photon uncaging is of special interest in epilepsy research, as imbalances between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission have been implicated in the pathogenesis of the disorder [27; 28; 74]. Uncaging is used in four of the studies included in this review. [PERSON] and [PERSON] [62] performed glutamate uncaging in dendrites of granule cells, whereas [PERSON] et al. [53] and [PERSON] et al. [54] used uncaging to trigger astrocytic [Ca2+] signaling and glutamatergic gliotransmission. The findings presented in the latter two publications were groundbreaking and paved the way for subsequent research into the role of astrocytes and astrocytic transformation in epilepsy. As such, the use of 2 PI directly and unequivocally contributed to our understanding of a novel aspect of epilepsy [72]. 2P-FLIM was used in both papers on neurotabolism. Fluorescence decay profiles of intrinsic NADH were measured in vivo surrounding seizures and induced metabolic inhibition. This led to imaging and confirmation of the hypothesis that NADH binding and metabolic capacity are significantly impaired following focal seizure activity [65; 66]. One paper successfully applied 2P-FLIM to image intrinsic NADH in living human brain tissue [66]. Furthermore, 2P-FLIM was used to identify the lifetime of substances, such as [Ca2+] and pO2, providing interesting information on quantified fluctuations of concentration over time [57; 58] while also allowing for identification and quantification of lipofuscin in the cerebral vasculature [21]. Finally, in 20 of the 38 included studies, 2 PI was used to image animal brains in vivo.