Fluorescence spectra and microscopic imaging of porphyrins in single cells and tissues

Fluorescence spectra of photosensitizing porphyrins in single cells and tissues were measured using advanced microscopic and fibre-optic techniques. The porphyrin emission bands at 620–700 nm were superposed by autofluorescence of cells and tissues, showing a broad maximum around 520 nm and some lower emission in the red part of the spectrum. To differentiate between these contributions, ‘red’ and ‘green’ spectral ranges were selected where autofluorescence had the same intensity. This selection was used for microscopic imaging to detect porphyrin distributions in tissues by subtraction of the intensity patterns of integral fluorescence-measured in the range of 590–800 nm—and autofluorescence—determined at 520–560 nm. The fluorescence intensities were measured and quantitated in squamous cell carcinomas of Syrian hamsters and in subcutaneously induced inflammations of Wistar rats. Due to quenching or re-absorption of the green fluorescence light in blood vessels, the method was not appropriate for porphyrin detection in vascular systems.