Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements
of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have
been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to
depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular
location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the
depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in
human skin.
Two-photon and confocal microscopy can be used to study the absorption of fluorescent compounds in tissue e.g. for
evaluation of topical drug delivery systems. Images of drug distribution at different depth in the skin with relatively
high spatial resolution are obtained. However, presented results are often static images from one single time point after
topical application.
We present an online diffusion cell with optical access allowing for temporal imaging of skin penetration and
measurement of percutaneous absorption in vitro. The temporal imaging cell (TIC) is adopted for both two-photon and
confocal microscopy. In this study the TIC has been used to visualize the change in absorption of Rhodamine B with
time at different depth in human epidermis using two-photon microscopy. Imaging showing the penetration of
Rhodamine B with time are presented together with transepidermal fluxes measured in TIC's and in traditional diffusion
cells.
In conclusion we have shown that the TIC is a promising tool for temporal studies of the absorption of fluorescent
compounds in tissue. E.g. sample preparation is held to a minimum prior to imaging, improved detection and resolution
in viable epidermis is achieved by imaging from the dorsal side of skin and the possibility to simultaneously analyze the
acceptor liquid increases the information available from each experiment.
The two-photon excitation point spread function (TPE-PSF) has been measured in human skin in vitro in order to
examine the optical resolution. This has been done by injecting fluorescent subresolution beads in skin samples
using a syringe. The beads were imaged at different depths and the full width at half maximum (FWHM) of the
TPE-PSF in the lateral and axial direction were measured from the intensity profile of the emission. The
experimentally obtained values of the PSF widths were larger than calculated values. Both the lateral FWHM and
the axial FWHM were broadened as a function of depth but the increase was stronger in the axial direction. This
indicates that the optical properties of the skin have a more pronounced effect of the resolution in the axial
direction.
For successful uptake and distribution of drugs from transdermal formulations, it is important to understand the skin
barrier function. Innovative advances in modern microscopy have provided valuable tools to study the interaction
between the skin and xenobiotics. Two-photon microscopy (TPM) allows non-invasive visualization of fluorescent
compounds in the skin. The advantages of TPM over conventional confocal microscopy are better light penetration into
highly scattering and absorbing tissue such as human skin, improved detection efficiency, limited out of focus
photobleaching and reduced phototoxic effects.
We present TPM as an alternative non-invasive in vitro method to study chemical penetration enhancement of
fluorescent model drugs. The permeability of sulforhodamine B (SRB) through human epidermis was measured with
vertical diffusion cells. The absorption was visualized using TPM after 24 h passive diffusion. We have evaluated
variations in physicochemical parameters controlling dermal drug uptake induced by the penetration enhancer oleic acid
according to methods previously described by Yu et al. Optical sectioning by TPM was compared with cryosectioning.
Oleic acid significantly increased penetration of sulforhodamine. TPM images demonstrate a four-fold increase in the
partition coefficient. In addition, a six-fold increase in the concentration gradient was found over stratum corneum.
Better light penetration and detection efficiency increase maximum imaging depth in TPM compared to conventional
confocal microscopy, however loss of signal due to scattering and absorption is still significant and will affect
distribution profiles generated by optical sectioning. A true concentration profile cannot be established without better
knowledge about signal losses in the skin.
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