The Kollias legacy: Skin autofluorescence and beyond.

In a paper published at the J Invest Dermatol in 1998 Nik Kollias and coworkers described distinct changes in skin native fluorescence associated with skin aging and photoaging, using in vivo fluorescence excitation spectroscopy. The assignment of the 295 nm band to tryptophan fluorescence had a profound significance influencing many later studies from multiple groups. The reproducible changes in skin native fluorescence suggested that aging causes predictable alterations in both the epidermis and the dermis, whereas chronic UV exposure induces the appearance of new fluorophores. This seminal, but insufficiently widely appreciated work deserves re-examination as it points to important horizons in future experimental dermatology, such as cancer diagnostics, diabetes, wound healing, and understanding skin aging and photoaging mechanisms.

Non-invasive assessment of tryptophan fluorescence and confocal microscopy provide information on skin barrier repair dynamics beyond TEWL.

The stratum corneum (SC) serves a primary function of skin barrier and understanding the kinetics of SC formation may provide great insight for skin diagnosis and evaluation of therapies. Besides trans-epidermal water loss (TEWL), few methods have been characterized to assess skin barrier non-invasively in vivo, particularly for dynamic measurements on the same specimen over time. The objective of this study was to characterize alternative non-invasive methods to evaluate the dynamic processes involved in the recovery of normal human SC after total removal. TEWL, tryptophan fluorescence and reflectance confocal microscopy (RCM) were used to determine skin barrier function, cell turnover and epidermal morphology over a period of 10 days after total removal of the SC by tape stripping. The results show a biphasic recovery of TEWL over time, which contrasted with a linear increase of 2.3 µm/day in SC thickness. Tryptophan assessment of cell turnover also demonstrated a biphasic pattern attaining a maximum three to four times the levels of the control site 3 days after injury that slowly returned to baseline and displayed great correlation (R(2)  > 0.95) to viable epidermis thickness that also achieved a maximum about 3 days after injury with an approximate increase of 55%. When plotting the change of TEWL versus SC thickness, a single exponential function is observed [Δ-TEWL = 55 exp (-0.157×)] which contrasts with other proposed models. These methods were able to present rates for SC recovery processes beyond skin barrier (TEWL) that may provide new insights on kinetics of barrier formation for evaluation of skin conditions and treatments.

Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence.

Optically monitoring the expression of green fluorescent protein (GFP) in the cartilage underlying the skin of a mouse allows tracking the expression of the chondrocyte phenotype. This paper considers how confocal microscopy with spectral detection can sense GFP fluorescence in the cartilage despite light scattering and collagen autofluorescence from the overlying skin. An in vivo experiment tested the abilities of a topical optical fiber measurement and a confocal microscope measurement to detect GFP in cartilage under the skin versus the collagen autofluorescence. An ex vivo experiment tested the ability of a confocal microscope without and with its pinhole to detect a fluorescent microsphere underneath an ex vivo skin layer versus the collagen autofluorescence. In both systems, spectroscopic detection followed by linear analysis allowed spectral discrimination of collagen autofluorescence (M(C)) and the subdermal green fluorescence (M(G)) due to either GFP or the microsphere. Contrast was defined as M(G)/(M(G)+M(C)). The in vivo contrast for GFP using optical fiber and confocal measurements was 0.16 and 0.92, respectively. The ex vivo contrast for a fluorescent microsphere using a confocal system without and with a pinhole was 0.13 and 0.48, respectively. The study demonstrates that a topical optical fiber measurement is affected by collagen autofluorescence, while a confocal microscope can detect subdermal fluorescence while rejecting collagen autofluorescence.

Skin responses to micro scale field size of solar-simulated radiation--preliminary evaluation by reflectance confocal microscopy in vivo.

Erythema and pigment responses of human skin following an acute exposure to ultraviolet radiation (UVR) are frequently used to determine the photosensitivity of the skin. In this study we investigated the responses of the skin to a micro-scale area of UVR exposure (MiR) and compared the responses to a macro-scale area of exposure (MaR). Ten human volunteers were tested with solar-simulated radiation on their upper arm or back using a beam size of 8 mm and 0.2 mm in diameter. The fluence required to produce a minimally perceptible erythema (MED) using the MiR was found to be higher than that for the MaR. The erythema response extended beyond the exposed area and this became pronounced when the beam size was microscopic. Reflectance confocal microscopy in vivo revealed that MiR induced cellular alterations within a confined area of smaller dimensions than the area of exposure. Pigment responses were confined within the areas of cellular damage. The erythema expression of exposed skin recovered faster for the sites receiving MiR even when the applied fluence was higher than the MED for the MaR. Through the use of MiR we were able to visualize spatially dissimilar skin responses of erythema and pigmentation suggesting different cellular mechanisms.

Interpreting diffuse reflectance for in vivo skin reactions in terms of chromophores.

The measurement and quantification of skin reactions to insults involves certain assumptions about the relation between intensity of color appearance of the skin and the concentration of endogenous chromophores. The underlying assumption is that the Beer-Lambert law is obeyed, i.e., that a linear relation exists between the absorbance and the concentration of each chromophore and that the total absorbance is the linear superposition of the contributions of each chromophore. In this paper the authors compiled the results from a number of interventions on human skin that result in changes in its appearance and small deviations from the homeostatic state, where the results may be accounted for by a single or multiple chromophores. The validity of the assumptions is found to hold for a limited range of responses. The biological constraints need to be considered in certain cases because as we move away from the homeostatic state, complex biological processes are induced.

Simultaneous assessment of pulsating and total blood in inflammatory skin lesions using functional diffuse reflectance spectroscopy in the visible range.

We present a simple and cost-effective optical technique for the simultaneous assessment of pulsating and total blood noninvasively in an inflammatory skin lesion. Acquisitions of diffuse reflectance spectra in the visible range at 6 Hz are used to trace the oscillating components of reflectance. Measurements on erythematous lesions from a UV insult show slow changing signal at about 0.1 Hz and heart-driven regular oscillations at about 1 Hz simultaneously. The results demonstrate the potential of the technique in monitoring both pulsating and steady components of the blood in inflammatory lesions of the skin.

Collection efficiency of a single optical fiber in turbid media.

If a single optical fiber is used for both delivery and collection of light, two major factors affect the measurement of collected light: (1) the light transport in the medium that describes the amount of light that returns to the fiber and (2) the light coupling to the optical fiber that depends on the angular distribution of photons entering the fiber. We focus on the importance of the latter factor and describe how the efficiency of the coupling depends on the optical properties of the medium. For highly scattering tissues, the efficiency is well predicted by the numerical aperture (NA) of the fiber. For lower scattering, such as in soft tissues, photons arrive at the fiber from deeper depths, and the coupling efficiency could increase twofold to threefold above that predicted by the NA.