Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

An example of multichannel FLIM data from endogenous fluorophores at the stratum granulosum level in human epidermis, recorded in three wavelength channels (350–450 nm, 450–515 nm, 515–620 nm) with excitation at 740 nm. The lower panel shows decay curves from a selected pixel fitted with a bi-exponential model. The three lifetime images in the upper panel show, from left to right, mainly NAD(P)H, both NAD(P)H and FAD and FAD only. The weak FAD signal and lack of cellular definition seen in the right FAD image indicates that 740 nm is not the optimal excitation wavelength for FAD. New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.