Analysis of Radachlorin localization in living cells by fluorescence lifetime imaging microscopy

PDT-Induced Variations of Radachlorin Fluorescence Lifetime in Living Cells In Vitro

“…Typical variations of this parameter in HeLa and A549 cells amounted to ±0.3 ns and ±0.4 ns, respectively. The diversity of the mean lifetime of Radachlorin fluorescence could be due to slightly different amounts of the photosensitizer accumulated in the lysosomes and cytoplasm of the cells at different stages of the cell cycle and different pH levels of intracellular media in them, since the acidity of the surrounding microenvironment can significantly affect Radachlorin fluorescence lifetime [24]. Despite the diversity of the mean fluorescence lifetime among individual cells, the applied experimental protocol of consecutive monitoring and analysis of the same set of microscopic fields of view allowed robust distinguishing of variations in the fluorescence properties of the intracellular photosensitizer.…”

“…We have recently shown that time-resolved fluorescence spectroscopy using Radachlorin allows assessing variations in microenvironmental acidity, polarity, and viscosity [24,36,44]. The FLIM-assisted imaging provided data on the PS uptake, concentration, and fluorescence quantum yield in cells and tissues [24]. In this study, we present an analysis of changes in the fluorescence characteristics of Radachlorin in cells in vitro as a result of photodynamic treatment.…”

“…Another common approach that can be used for real-time PDT dosimetry relies on the monitoring of photosensitizer fluorescence intensity [19][20][21]. However, the accuracy of this approach is quite low due to the non-uniformity of the absorption and scattering properties of tissues [22,23], as well as the potential dependence of fluorophore fluorescence intensity on microenvironment properties, acidity in particular [24]. Nevertheless, an analysis of the distributions of photosensitizer fluorescence intensity is applied for the intraoperative delimitation of tumor boundaries [25,26].…”

“…The analysis of time-resolved fluorescence signals was applied for characterization of various PSs in solutions [36], cells in vitro [37,38], and tissues in vivo [39,40]. The utilization of fluorescence lifetime spectroscopy and microscopy allowed for the analysis of photoproduct formation [41,42], the intracellular localization of PS molecules [24,38], and their internalization in an aggregated form [43].…”

“…We have recently shown that time-resolved fluorescence spectroscopy using Radachlorin allows assessing variations in microenvironmental acidity, polarity, and viscosity [24,36,44]. The FLIM-assisted imaging provided data on the PS uptake, concentration, and fluorescence quantum yield in cells and tissues [24].…”