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.

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.

Approach: Mice were intravenously injected with 2.5 mg/kg of photosensitizer aluminum(III) phthalocyanine tetrasulfonate (AlPcS₄) 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.

Results: Delayed emission from AlPcS₄ 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  /  cm². In addition, lifetime images of AlPcS₄ 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.

Conclusions: Imaging of SOFDF in vivo in mouse tumor during PDT with AlPcS₄ 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.

Significance: While clinical treatment of actinic keratosis by photodynamic therapy (PDT) is widely practiced, there is a well-known variability in response, primarily caused by heterogeneous accumulation of the photosensitizer protoporphyrin IX (PpIX) between patients and between lesions, but measurement of this is rarely done.

Aim: Develop a smartphone-based fluorescence imager for simple quantitative photography of the lesions and their PpIX levels that can be used in a new clinical workflow to guide the reliability of aminolevulinic acid (ALA) application for improved lesion clearance.

Approach: The smartphone fluorescence imager uses an iPhone and a custom iOS application for image acquisition, a 3D-printed base for measurement standardization, an emission filter for PpIX fluorescence isolation, and a 405-nm LED ring for optical excitation. System performance was tested to ensure measurement reproducibility and the ability to capture photosensitizer accumulation and photobleaching in pre-clinical and clinical settings.

Results: PpIX fluorescence signal from tissue-simulating phantoms showed linear sensitivity in the 0.01 to 2.0 μM range. Murine studies with Ameluz^® aminolevulinic acid (ALA) gel and initial human testing with Levulan^® ALA cream verified that in-vivo imaging was feasible, including that PpIX production over 1 h is easily captured and that photobleaching from the light treatment could be quantified.

Conclusions: The presented device is the first quantitative wide-field fluorescence imaging system for PDT dosimetry designed for clinical skin use and for maximal ease of translation into clinical workflow. The results lay the foundation for using the system in clinical studies to establish treatment thresholds for the individualization of PDT treatment.