KEYWORDS: Optical simulations, Beam shaping, Skin, Multiphoton microscopy, Point spread functions, Light scattering, Luminescence, Microscopes, Signal attenuation, Monte Carlo methods
Multiphoton fluorescence microscopy (MPM) is a method for high resolution, non-invasive investigations of biological tissue. The aim of introducing an annular shaped laser beam is to reduce the ouf-of-focus generated background signal improving imaging of light scattering tissue such as human skin. Simulations show that 50% of the beam radius can be blocked, while preserving the shape of the point spread function. Initial experiments performed on a phantom consisting of fluorescein and fluorescent beads embedded in agar by using a custom built MPM-set up show that by introducing a simple beam blocker to create an annular beam, the background signal is reduced with approximately 5%. Future work will include optimizing the set up, and creating phantoms with more light scattering properties.
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.
The complex structure of skin represents an effective barrier against external environmental factors, as for example,
different chemical and biochemical compounds, yeast, bacterial and viral infections. However, this impermeability
prevents efficient transdermal drug delivery which limits the number of drugs that are able to penetrate the skin
efficiently. Current trends in drug application through skin focus on the design and use of nanocarriers for transport of
active compounds. The transport systems applied so far have several drawbacks, as they often have low payload, high
toxicity, a limited variability of inclusion molecules, or long degradation times. The aim of these current studies is to
investigate novel topical drug delivery systems, e.g. nanocarriers based on cyclic oligosaccharides - cyclodextrins (CD)
or iron (III)-based metal-organic frameworks (MOF). Earlier studies on cell cultures imply that these drug nanocarriers
show promising characteristics compared to other drug delivery systems.
In our studies, we use two-photon microscopy to investigate the ability of the nanocarriers to deliver compounds through
ex-vivo skin samples. Using near infrared light for excitation in the so called optical window of skin allows deep-tissue
visualization of drug distribution and localization. In addition, it is possible to employ two-photon based fluorescence
correlation spectroscopy for quantitative analysis of drug distribution and concentrations in different cell layers.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.