Ategy to induce deep tissue phototoxicity is to perform repeated PDT or metronomic [39] PDT (slow infusion of PS and low dose light). In the realm of repeated PDT, studies have shown that fractionated PDT (i.e., PDT repeated with a prefixed time interval in one therapy session) induced necrosis to a depth 3 times greater than PDT alone [40]. In addition to affording a better treatment response profile, this PDT design also increases the feasibility of deep tissue PDT because it may allow for continuous accumulation of PSs at the treatment site, i.e., the first series of irradiation of PpIX in ALA-based PDT will lead to photobleaching of the PpIX and the time gap between irradiations will allow for resynthesis of PpIX to occur at the treatment site. The amount of PpIX reaccumulated at the treated site is demonstrated to be a function of the fluence rate of the first PDT dose [23, 41]. These studies indicate that clever PS delivery strategies together with appropriate light illumination strategies could lend themselves to more efficacious deep tissue PDT.tumors impacts PS uptake, thereby further altering the tissue optical properties. Understanding the spatial distribution of light in BFA site lesions and personalizing design strategies such as the placement of fiber optic probes or adjusting fluence rate based on real-time feedback on lesion properties (PS concentration, photobleaching, oxygenation content, etc.) is of the utmost importance to achieve predictable treatment outcomes from PDT. For example, Zhou et al demonstrated that personalizing the light dose based on pre-treatment measurements of the PS concentration within the lesion significantly reduced variability in treatment response [43]. Another important factor determining PDT efficacy is the PS-light-interval, wherein dosimetry and treatment planning can become complicated when considering damage to only the vascular compartment of the lesions and not to the surrounding tissue [44]. Fluorescence imaging has traditionally played a major role in PDT dosimetry by evaluating PS fluorescence and photobleaching [3, 15]; however, its XAV-939 custom synthesis penetration depth is limited and makes it difficult to gauge deeply-situated untreated regions. Other deep-tissue optical imaging techniques such as photoacoustic imaging [45] or diffuse optical imaging techniques [46] are currently being evaluated in several studies to understand the role of oxygen in PDT efficacy. In our recent studies, we showed that regions within the tumor that did not have complete vascular shutdown (i.e., no reduction in blood oxygen saturation) regrew post PDT [47]. Fig. 4 showcases an example of untreated regions within the subcutaneous tumor (xenograft with U87 glioblastoma cells) where there was no hypoxia due to vascular shutdown. Specifically, an ultrasound image (tumor structure), photoacoustic image (oxygen saturation), and immunofluorescence image (vasculature in green and hypoxic regions in red) of aImage-guided dosimetry and treatment design for deep-tissue PDTTissue optical properties play a dominant role in determining the depth of the treatment zone during PDT [2, 42] and moreover, due to the variable vascular network and microenvironment in pathologies such as cancer, there is significant interand intra-lesion heterogeneity in treatment response. For example, the heterogeneous vascular network inFigure 4: Utility of deep-tissue photoacoustic imaging to monitor PDT efficacy. The ultrasound image demarcates the location and.Ategy to induce deep tissue phototoxicity is to perform repeated PDT or metronomic [39] PDT (slow infusion of PS and low dose light). In the realm of repeated PDT, studies have shown that fractionated PDT (i.e., PDT repeated with a prefixed time interval in one therapy session) induced necrosis to a depth 3 times greater than PDT alone [40]. In addition to affording a better treatment response profile, this PDT design also increases the feasibility of deep tissue PDT because it may allow for continuous accumulation of PSs at the treatment site, i.e., the first series of irradiation of PpIX in ALA-based PDT will lead to photobleaching of the PpIX and the time gap between irradiations will allow for resynthesis of PpIX to occur at the treatment site. The amount of PpIX reaccumulated at the treated site is demonstrated to be a function of the fluence rate of the first PDT dose [23, 41]. These studies indicate that clever PS delivery strategies together with appropriate light illumination strategies could lend themselves to more efficacious deep tissue PDT.tumors impacts PS uptake, thereby further altering the tissue optical properties. Understanding the spatial distribution of light in lesions and personalizing design strategies such as the placement of fiber optic probes or adjusting fluence rate based on real-time feedback on lesion properties (PS concentration, photobleaching, oxygenation content, etc.) is of the utmost importance to achieve predictable treatment outcomes from PDT. For example, Zhou et al demonstrated that personalizing the light dose based on pre-treatment measurements of the PS concentration within the lesion significantly reduced variability in treatment response [43]. Another important factor determining PDT efficacy is the PS-light-interval, wherein dosimetry and treatment planning can become complicated when considering damage to only the vascular compartment of the lesions and not to the surrounding tissue [44]. Fluorescence imaging has traditionally played a major role in PDT dosimetry by evaluating PS fluorescence and photobleaching [3, 15]; however, its penetration depth is limited and makes it difficult to gauge deeply-situated untreated regions. Other deep-tissue optical imaging techniques such as photoacoustic imaging [45] or diffuse optical imaging techniques [46] are currently being evaluated in several studies to understand the role of oxygen in PDT efficacy. In our recent studies, we showed that regions within the tumor that did not have complete vascular shutdown (i.e., no reduction in blood oxygen saturation) regrew post PDT [47]. Fig. 4 showcases an example of untreated regions within the subcutaneous tumor (xenograft with U87 glioblastoma cells) where there was no hypoxia due to vascular shutdown. Specifically, an ultrasound image (tumor structure), photoacoustic image (oxygen saturation), and immunofluorescence image (vasculature in green and hypoxic regions in red) of aImage-guided dosimetry and treatment design for deep-tissue PDTTissue optical properties play a dominant role in determining the depth of the treatment zone during PDT [2, 42] and moreover, due to the variable vascular network and microenvironment in pathologies such as cancer, there is significant interand intra-lesion heterogeneity in treatment response. For example, the heterogeneous vascular network inFigure 4: Utility of deep-tissue photoacoustic imaging to monitor PDT efficacy. The ultrasound image demarcates the location and.