Multiphoton and FLIM imaging in quantifying ex vivo and in vivo body organ kinetics of solutes

Traditionally, destructive sampling and analysis are used to determine the fate, kinetics and effects of exogenous materials in the body. Minimally invasive confocal and multiphoton microscopy (MPM) in 3D space over in time to deep tissue depths has enabled us to quantify endogenous fluorescent species in the body as well as exogenous fluorescent molecules, cells and nanoparticles that have been administered into the body and/or are applied to the skin, kidney and liver ex vivo and in vivo. Of particular importance has been the ability to get specificity in drug, metabolite and endogenous solute measurement in tissues in vivo by using specific spectral excitation and emission wavelengths, the use of fluorescence lifetime and the measurement of fluorescence anisotropy. We have applied MPM to characterise physiologically based pharmacokinetics of solutes, mesenchymal stem cells and nanoparticles in various organs. More recently, we have used MPM to examine stem cell and nanoparticle – tissue interactions directly in acute liver and kidney injury models, tumor models and inflammatory models. MPM has also been used to measure changes in the redox state of cells, as well the use of photochemical probes to measure adverse biochemical events such as the formation of reactive oxygen species. Sun-induced skin damage, with its sequelae of photoaging, actinic keratosis and various skin cancers is a particular issue for many of us in subtropical and temperate climates. Our group has therefore also used MPM to quantify the metabolic changes seen in melanoma lesions, the safety of nanoparticle sunscreens, whose use may prevent these lesions, and to aid in the mechanistic and regulatory evaluation of topical product efficacy, bioequivalence and safety. In conclusion, MPM fluorescence lifetime imaging microscopy (FLIM) is a promising technology to aid in product characterization and development as well as in the translational diagnosis of skin related pathologies in the clinic.