KEYWORDS: Skin, In vivo imaging, Microscopy, Ultraviolet radiation, Imaging systems, Confocal microscopy, Two photon imaging, Solar radiation, Biopsy, Biological research
Background: Serial analysis of cellular dynamics over time offers new insights into human skin responses to solar radiation. However, most of the previous studies are based on biopsy ex vivo analysis approaches that preclude the monitoring of the same cells and sites over time. Optical in vivo microscopy enables the possibility of real-time live cell imaging. Here we report a robust non-invasive method to achieve repeated access to the same micro-location over a long period with unprecedented precision.
Methods: The technique is based on a temporary “surface marker” as landmark to help locate the same cells or microstructures between imaging sessions. At baseline, the region-of-interest (ROI) is determined and imaged. At follow up sessions, the ROI can be automatically located. Using this method, we precisely revisited the same cells in human skin after UVB radiation over two weeks. Skin microscopic responses was studied with a multimodality in vivo microscopy system capable of co-registered video rate reflectance confocal microscopy (RCM) imaging, two-photon fluorescence (TPF) imaging and second harmonic generation (SHG) imaging.
Results: The quantitative analysis of TPF signal revealed that melanin distribution pattern changed with time after UVB exposure, suggesting that melanin migrates towards the skin surface. Blood flow was monitored in the same capillary over two weeks. Multimodal analyses enabled accurate calculation of viable epidermis, stratum corneum thickness and cell density variations over time, demonstrating the time points of tissue edema and cell proliferation.
Colorimetry has been used as an objective measure of perceived skin color by human eye to document and
score physiological responses of the skin from external insults. CIE color space values (L*, a* and b*) are the
most commonly used parameters to correlate visually perceived color attributes such as L* for pigment, a* for
erythema, and b* for sallowness of the skin. In this study, we investigated the relation of Lab color scale to the
amount of major skin chromophores (oxy-, deoxyhemoglobin and melanin) calculated from diffuse reflectance
spectroscopy. Thirty two healthy human subjects with ages from 20 to 70 years old, skin types I-VI, were
recruited for the study. DRS and colorimetry measurements were taken from the left and right cheeks, and
on the right upper inner arm. The melanin content calculated from 630-700 nm range of DRS measurements
was shown to correlate with the lightness of skin (L*) for most skin types. For subjects with medium-to-light
complexion, melanin measured at the blue part spectrum and hemoglobin interfered on the relation of lightness
of the skin color to the melanin content. The sallowness of the skin that is quantified by the melanin contribution
at the blue part spectrum of DRS was found to be related to b* scale. This study demonstrates the importance
of documenting skin color by assessing individual skin chromophores with diffuse reflectance spectroscopy, in
comparison to colorimetry assessment.
The minimal erythema dose induced by solar-simulated radiation is a useful measure of UV sensitivity of
skin. Most skin phototests have been conducted by projecting a flat field of UV radiation onto the skin in
an area greater than 15 cm × 15 cm with an increment of radiation doses. In this study, we investigated the
responses of human skin to solar-simulated radiation of different field sizes. Twelve human subjects of skin
phototype I-IV were exposed to solar-simulated radiation (SSR) on their upper inner arm or on their lower
back with a series of doses in increments of 20% in order to determine the threshold dose to induce a minimal
perceptible erythema response (MED). Each dose was delivered with a liquid light guide (8 mm diameter on
the back or 6 mm on the upper inner arm) and with quartz optical fibers of 200 μm diameter. The resulting
skin responses were evaluated visually and investigated with a reflectance confocal microscope and imaging.
The erythema response to the microscopic challenge was always diffuse with no clear boundaries extending to
several times the exposed site diameter at doses greater than 2 MED. The skin returned to normal appearance
from the microscopic challenge after two weeks of exposure while change in appearance for the larger areas
persisted for several weeks to months. This new modality of testing provides the possibility to study skin at
the microscopic level with a rapid recovery following challenge.
The stratum corneum (SC) serves a primary function of skin barrier and its maintenance is vital for the existence of
terrestrial life. Few studies have been performed for evaluation of human SC repair in vivo, non-invasively. In the
present study tape stripping was performed on the arms and legs of seven volunteers until all the SC was removed. The
injured site and a control adjacent site were measured over a period of 10 days after the injury to assess functionality and
repair. Transepidermal water loss (TEWL), tryptophan fluorescence and reflectance confocal microscopy were used to
determine permeability of the skin barrier, cell turnover and epidermis morphology, respectively. The results show an
exponential rate of recovery for the skin permeability (TEWL) which contrasted with a linear increase in the thickness of
the SC as determined by confocal microscopy. Cell turnover increased rapidly immediately after the injury to 2.5 times
the levels of the control site, attaining a maximum of 3.5-4 times greater levels after three days and slowly returned to
baseline levels after the ten days. Correlation of the cell turnover to the thickness of the viable epidermis was observed
and further studies are under way to interpret these results.
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.
Measurement of cutaneous microcirculation is of great importance for clinical evaluations as many biological processes (i.e. inflammation) activate superficial vessels. Diffuse Reflectance Spectroscopy (DRS) is a widely used method to assess cutaneous microcirculation since it is based on the spectral characteristics of skin chromophores, particularly the strong absorption bands of blood in the visible spectral range. However, the DRS system only provides a snapshot of the blood content in tissue from the reflectance detected at a single exposure time, and it fails to demonstrate dynamic changes of blood flow inside the tissues. In the present study, a prototype system for functional DRS (fDRS) has been developed for the sequential acquisition of multiple reflectance spectra in the visible range at sub-second intervals. Twelve healthy subjects with skin phototype I-III were recruited for a UV skin phototest where the subject's back was exposed to an increasing
dose of solar-simulated radiation at a maximum of 3 minimum erythema dose (MED). Evaluations included conventional DRS, functional DRS, and a laser Doppler flowmeter/imaging system. In a frequency analysis
results, there were two distinctive frequency components. A low-frequency component was found near 0.03-0.1 Hz, and another high-frequency component near 0.9-1.2 Hz which is synchronous with heart pulsations.
The magnitude of the high-frequency and the steady-state components of the fDRS signal increased with
increase in exposure dose. These results demonstrate the potential of this technique for noninvasive
assessment of cutaneous microcirculation.
The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular
the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris.
Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for
production of heme.
In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum
and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of
a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A
computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm
then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used
to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The
resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of
healthy subjects. The results show great variability on the concentration of these porphyrins and further studies
are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.
The use of perturbation and differential Monte Carlo (pMC/dMC) methods in conjunction with nonlinear optimization algorithms were proposed recently as a means to solve inverse photon migration problems in regionwise heterogeneous turbid media. We demonstrate the application of pMC/dMC methods for the recovery of optical properties in a two-layer extended epithelial tissue model from experimental measurements of spatially resolved diffuse reflectance. The results demonstrate that pMC/dMC methods provide a rapid and accurate approach to solve two-region inverse photon migration problems in the transport regime, that is, on spatial scales smaller than a transport mean free path and in media where optical scattering need not dominate absorption. The pMC/dMC approach is found to be effective over a broad range of absorption (50 to 400%) and scattering (70 to 130%) perturbations. The recovery of optical properties from spatially resolved diffuse reflectance measurements is examined for different sets of source-detector separation. These results provide some guidance for the design of compact fiber-based probes to determine and isolate optical properties from both epithelial and stromal layers of superficial tissues.
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