Imaging assessment of photosensitizer emission induced by radionuclide-derived Cherenkov radiation using charge-coupled device optical imaging and long-pass filters.

Background: Radionuclides produce Cherenkov radiation (CR), which can potentially activate photosensitizers (PSs) in phototherapy. Several groups have studied Cherenkov energy transfer to PSs using optical imaging; however, cost-effectively identifying whether PSs are excited by radionuclide-derived CR and detecting fluorescence emission from excited PSs remain a challenge. Many laboratories face the need for expensive dedicated equipment.

Aim: To cost-effectively confirm whether PSs are excited by radionuclide-derived CR and distinguish fluorescence emission from excited PSs.

Methods: The absorbance and fluorescence spectra of PSs were measured using a microplate reader and fluorescence spectrometer to examine the photo-physical properties of PSs. To mitigate the need for expensive dedicated equipment and achieve the aim of the study, we developed a method that utilizes a charge-coupled device optical imaging system and appropriate long-pass filters of different wavelengths (manual sequential application of long-pass filters of 515, 580, 645, 700, 750, and 800 nm). Tetrakis (4-carboxyphenyl) porphyrin (TCPP) was utilized as a model PS. Different doses of copper-64 (⁶⁴CuCl₂) (4, 2, and 1 mCi) were used as CR-producing radionuclides. Imaging and data acquisition were performed 0.5 h after sample preparation. Differential image analysis was conducted by using ImageJ software (National Institutes of Health) to visually evaluate TCPP fluorescence.

Results: The maximum absorbance of TCPP was at 390-430 nm, and the emission peak was at 670 nm. The CR and CR-induced TCPP emissions were observed using the optical imaging system and the high-transmittance long-pass filters described above. The emission spectra of TCPP with a peak in the 645-700 nm window were obtained by calculation and subtraction based on the serial signal intensity (total flux) difference between ⁶⁴CuCl₂ + TCPP and ⁶⁴CuCl₂. Moreover, the differential fluorescence images of TCPP were obtained by subtracting the ⁶⁴CuCl₂ image from the ⁶⁴CuCl₂ + TCPP image. The experimental results considering different ⁶⁴CuCl₂ doses showed a dose-dependent trend. These results demonstrate that a bioluminescence imaging device coupled with different long-pass filters and subtraction image processing can confirm the emission spectra and differential fluorescence images of CR-induced TCPP.

Conclusion: This simple method identifies the PS fluorescence emission generated by radionuclide-derived CR and can contribute to accelerating the development of Cherenkov energy transfer imaging and the discovery of new PSs.