Extracellular skin structures in human skin are impaired during intrinsic and extrinsic aging. Assessment of these dermal changes is conducted by subjective clinical evaluation and histological and molecular analysis. We aimed to develop a new parameter for the noninvasive quantitative determination of dermal skin alterations utilizing the high-resolution three-dimensional multiphoton laser scanning microscopy (MPLSM) technique. To quantify structural differences between chronically sun-exposed and sun-protected human skin, the respective collagen-specific second harmonic generation and the elastin-specific autofluorescence signals were recorded in young and elderly volunteers using the MPLSM technique. After image processing, the elastin-to-collagen ratio (ELCOR) was calculated. Results show that the ELCOR parameter of volar forearm skin significantly increases with age. For elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area is significantly augmented compared to the sun-protected upper arm area. Based on the MPLSM technology, we introduce the ELCOR parameter as a new means to quantify accurately age-associated alterations in the extracellular matrix.
There are visible changes during skin aging. In the extracellular matrix these changes referred to as intrinsic aging (skin areas not exposed to sunlight) and extrinsic aging can be measured using various methods, such as subjective clinical evaluation, histology and molecular analysis. In this study we developed a new parameter for the non-invasive quantitative determination of dermal skin aging utilizing a five-dimensional intravital tomography (5D-IVT). This device, also known as 5D - multi-photon laser scanning microscopy, is a powerful tool to investigate (photo)aging-associated
alterations in vivo.
Structural alterations in the dermis of extrinsically aged (chronically sun-exposed) and intrinsically aged (sun-protected) human skin were recorded utilizing the collagen-specific second harmonic generation (SHG) signal and the elastin-specific autofluorescence (AF) signal. Recording took place in young and elderly volunteers. The resulting images were processed in order to gain the elastin percentage and the collagen percentage per image. Then, the elastin - to - collagen ratio (ELCOR) was calculated. With respect to volar forearm skin, the ELCOR significantly increased with age. In elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area was significantly augmented compared with the sun-protected upper arm area.
Based on 5D-IVT we introduce the ELCOR as a new means to quantify age-associated alterations in the extracellular matrix of in vivo human skin. This novel parameter is compared to the currently
used "SHG to AF aging index" of the dermis (SAAID).
One of the major structural proteins in human skin is collagen. Collagen and its crosslinks are
essential for the mechanical stability of the skin. Looking at extrinsically aged human skin (photo
damaged skin) we find a decrease of mature collagen crosslinks. Immature crosslinks an indicator of
the collagen turnover are decreasing as well in extrinsically aged skin. Hence, we assume that a
certain range of mature and immature crosslinks reflect a 'good quality' of collagen in terms of
photoaging.
In this study we established in vitro models of reduced crosslinking. We found that reduced collagen
crosslinking resulted in a higher Second Harmonic Generation (SHG) intensity. Furthermore, we
found a higher fibril diameter after crosslink reduction without an increase in collagen concentration.
SHG is generated by a non-linear effect of femtosecond laser irradiation on collagen molecules. This
effect might be influenced by the interspaces of the collagen molecules within the collagen fibril.
From these findings the following hypothesis was introduced: reduced collagen crosslinking changes
the interspace of single collagen molecules within the collagen fibril resulting in an enhanced SHG
signal.
Furthermore, in this study the fluorescence lifetime (FLIM) of collagen fluorescence was found to
decrease in the in vitro models of reduced crosslinking. We speculate on possible mechanisms being
responsible for the decrease in lifetime.
Future in vivo measurements of the two parameters (SHG and FLIM) could lead to information
about the collagen crosslink status, and therefore the status of photoaging of the skin.
Multiphoton optical tomography or intravital tomography (IVT) provides non-invasive optical sectioning of biological
specimens, e.g. skin, with subcellular spatial resolution without any need of contrast agents. It can be used to distinguish
between normal and diseased tissue due to the differences in morphological appearance. Additional information beyond
morphology can be obtained by analyzing the collected fluorescence light spectroscopically and by means of its
fluorescence decay time. This is frequently termed spectral fluorescence lifetime imaging (SFLIM) or 5D-intravital
tomography (5D-IVT). Spectral and temporal resolution scales with the number of detection increments (i.e. spectral
channels and time bins). 5D-IVT enables us to detect new physiological parameters, however accompanied by a decrease
in intensity per channel. Moreover, the increase of data requests a higher need of software skills.
In this study we investigate and evaluate different technical modes of 5D-IVT with respect to their clinical relevance: (1)
a multichannel photomultiplier tube (PMT) array coupled to a diffraction grating, each channel being analyzed by timecorrelated
single photon counting (TCSPC), (2) three separate PMTs in spectral separation path using dichroic mirrors,
each channel being analyzed by TCSPC and (3) a single PMT TCSPC setup in combination with a high-resolution CCDspectrograph
for pointwise microspectroscopy.
New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in human skin. However, little is known about possible risks. In order to evaluate the risk, a “biological dosimeter” was used. We irradiated fresh human skin samples with both an fs-laser and a solar simulator UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at 800-nm wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation power using 10 horizontal scans every 5 microns. CPDs could be seen at 60-mW laser power and 5-µm step size and at 35-mW using 1-micron step width. Quantitative comparison of SSU-induced CPDs showed that the 60-mW laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The 1-micron irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.
During the last couple of years new imaging techniques using femtosecond lasers (fs-lasers) in the near infrared spectral range evolved for a variety of in vitro applications. We wanted to know, whether fs-lasers have a non-invasive imaging potential for in vivo applications for human skin. So far, little is known about possible risks of this irradiation type. To estimate the risk of irradiation damage in human skin we used a "biological dosimeter" in this investigation. We irradiated fresh human skin samples with both an fs-laser and a solar simulator (UV-source) for comparison. DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immuno-fluorescence image analysis. Various fs-irradiation regimes were evaluated differing in laser power and step width of horizontal irradiation scans.
When using 15 mW or 30 mW fs-laser power combined with horizontal irradiation scans applied every 5 mm in depth around the epidermal-dermal junction no induction of CPDs was found. However, induction of CPDs could be seen using 60 mW laser power and 5 μm step width. Narrowing the step width to 1 mm and using increasing laser power (up to 35 mW) from the surface of the skin to the epidermis led to CPD formation, too. Quantitative comparison of CPD production at various laser regimes with CPD production using a solar simulator was done. We could show that the number of CPDs formed by the 60 mW laser irradiation regime is comparable to an UV-irradiation giving 0.6 MED (minimal erythemal dose). The smaller step width laser irradiation regime (1 μm step width and up to 35 mW) was comparable to a UV-irradiation regime resulting in 0.45 MED.
Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.
Submicron surface-relief gratings were fabricated on fused silica by laser ablation with nanosecond (ns) pulses from a high-resolution F2-laser processing station. The grating relief was generated by imaging a transmission amplitude grating with a Schwarzschild objective of 25x demagnification. The chrome-coated CaF2 mask had been structured by laser ablation at 193 nm to form a line and space pattern of 20-μm period. The F2-laser generated gratings on fused silica were characterized by SEM, AFM and diffraction of a HeNe laser beam, yielding a grating period of 830 nm and a corrugation depth of 250 nm. Surface-relief gratings on optical materials are required for various applications such as grating demultiplexers for telecommunication components, light couplers for planar optical waveguides, Bragg reflectors, or alignment grooves for liquid crystals. Laser ablation is a rapid and flexible method to generate custom grating designs on a variety of materials.
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