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Significance: Singlet oxygen is a key cytotoxic agent in photodynamic therapy (PDT). As such, its imaging is highly desirable, but existing direct imaging methods are still limited by the exceptionally low yield of the luminescence signal. Singlet oxygen feedback delayed fluorescence (SOFDF) of the photosensitizer is a higher yield alternative for indirect measurement of this signal. &lt;p&gt; Aim: The aim was to explore feasibility of SOFDF imaging in vivo in tumor-bearing mice during PDT and investigate how SOFDF images can be transformed into images of singlet oxygen. In addition, we study whether lysosome permeabilization can be visualized through fluorescence lifetime.&lt;/p&gt; &lt;p&gt; Approach: Mice were intravenously injected with 2.5 mg/kg of photosensitizer aluminum(III) phthalocyanine tetrasulfonate (AlPcS 4 ) 20 h prior to experiments, having subcutaneous BxPC3 pancreas tumors. Time-resolved delayed fluorescence and prompt fluorescence (PF) were imaged using an intensified time-gated camera with 10-Hz pulsed laser excitation at 690 nm. &lt;/p&gt; &lt;p&gt; Results: Delayed emission from AlPcS 4 was detected with lifetimes 7 to 11  μs, which was attributed to SOFDF and shown to be oxygen-dependent. Singlet oxygen images were approximated by the ratio of SOFDF/PF at each pixel. SOFDF images of a good quality could be captured within several seconds with a radiant exposure of ∼20  mJ  /  cm 2 . In addition, lifetime images of AlPcS 4 PF in ns-time domain enabled us to visualize the event of lysosome permeabilization, as the lifetime increased from ∼4.7 to 5.2 ns. &lt;/p&gt; &lt;p&gt; Conclusions: Imaging of SOFDF in vivo in mouse tumor during PDT with AlPcS 4 is feasible, and it is a promising method for singlet molecular oxygen monitoring. Moreover, the time-gated approach also enables visualization of the lysosome permeabilization that alters the PF lifetime. &lt;/p&gt;

journal of biomedical optics

Imaging of singlet oxygen feedback delayed fluorescence and lysosome permeabilization in tumor in vivo during photodynamic therapy with aluminum phthalocyanine