Multiphoton fluorescence lifetime imaging (FLIM) is gaining ground as a non-invasive and very sensitive method in life sciences, and even as a clinical tool. First clinical devices employing FLIM are on the market, e.g. MPTflex. A hot topic is using metabolic imaging to investigate melanoma lesions (Fig.1). This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH) [1,2,3,4]. In another study, we investigated safety aspects of nanoparticle based sun screens. Multiphoton FLIM enables tracing of nanoparticles after application on the skin . Furthermore, in case of penetration into the viable epidermis metabolic imaging can be employed to investigate toxicity on skin cells .
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.
We describe the contribution of our in vivo multiphoton microscopy (MPM) studies over the last ten years with DermaInspect® (JenLab, Germany), a CE-certified medical tomograph based on detection of fluorescent biomolecules, to the assessment of possible penetration of nanoparticulate zinc oxide in sunscreen through human skin. At the time we started our work, there was a strong movement for the precautionary principle to be applied to the use of nanoparticles in consumer products due to a lack of knowledge. The combined application of different MPM modalities, including spectral imaging, fluorescence lifetime imaging, second harmonic fluorescence generation, and phosphorescence microscopy, has provided overwhelming evidence that nanoparticle zinc oxide particles do not penetrate human skin when applied to various skin types with a range of methods of topical sunscreen application. MPM has also been used to study the viable epidermal morphology and redox state in supporting the safe use of topical zinc oxide nanoparticles. The impact of this work is emphasized by the recent proposed rule by the United States FDA on Sunscreen Drug Products for Over-the-Counter Human Use, which listed only zinc oxide and titanium dioxide of the currently marketed products to be generally recognized as safe and effective.