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

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

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.

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.

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.

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.