Fluorescence microspectroscopy technique for the study of intracellular protoporphyrin IX dynamics

We describe a technique designed to monitor the fluorescence dynamics of photosensitizers used in photodynamic therapy (PDT) at micrometer-scale locations within individual formalin-fixed cells. The accumulation of protoporphyrin IX (PpIX) within keratinocytes and fibroblasts. following incubation with 5-aminolaevulinic acid (ALA), is shown to be dependent upon both incubation time and cell proliferation status. Also, the process of photobleaching within these cells is demonstrated via the depletion in PpIX fluorescence emission during exposure to 532 nm light. All spectra show a progressive reduction of the 634 nm PpIX peak, following a bi-exponential decay that is consistent with a singlet oxygen mediated process. The rate of photobleaching, when plotted as a function of light dose, increases with reduced incident laser power. The generation of the hydroxyaldehyde-chlorin photoproduct (photoprotoporphyrin), as monitored by the increase in fluorescence emission centered on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, PpIX fluorescence recovers during the dark period and there is an increase in bleaching rate at the onset of the second exposure. These results are qualitatively consistent with measurements performed in vivo, which demonstrate that the photodynamic dose is dependent upon fluence rate and oxygen status.