This PDF file contains the editorial “Special Section Guest Editorial: Optics of Human Skin” for JBO Vol. 9 Issue 02

Laser thermal injury and subsequent wound healing in organotypic, skin-equivalent tissue models were monitored using optical coherence tomography (OCT), multiphoton microscopy (MPM), and histopathology. The in vitro skin-equivalent raft tissue model was composed of dermis with type I collagen and fibroblast cells and epidermis of differentiated keratinocytes. Noninvasive optical imaging techniques were used for time-dependent, serial measurements of matrix destruction and reconstruction and compared with histopathology. The region of laser thermal injury was clearly delineated in OCT images by low signal intensity. High resolution MPM imaging using second-harmonic generation revealed alterations in collagen microstructure organization with subsequent matrix reconstruction. Fibroblast cell migration in response to injury was monitored by MPM using two-photon excited fluorescence. This study illustrates the complementary features of linear and nonlinear light–tissue interaction in intrinsic signal optical imaging and their use for noninvasive, serial monitoring of wound healing processes in biological tissues.

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.

We have proposed an optical probe that can be used to characterize the orientation of collagen fibers in human dermis. A specific probing ability for collagen results from the use of second-harmonic-generation (SHG) light induced by collagen molecules in the tissue. Based on the concept of collagen SHG light, a reflection-type polarization measurement system (named SHG polarimetry) with a probe light spot of 15 µm in diameter has been constructed, and the human reticular dermis has been measured using this system. Resultant data exhibit that the reticular dermis possesses approximately uniaxial orientation of the collagen fibers. Furthermore, we demonstrated a nondestructive measurement of the collagen orientation in the papillary dermis across an epidermis layer. For distribution measurement of the collagen fiber orientation in the reticular dermis, we have extended the SHG polarimetry to one- (1-D) and two-dimensional (2-D) measurement. By the macroscopic 2-D SHG polarimetry, we have observed that the orientation angle and organization degree of collagen fibers vary widely depending on the discrete probing positions in the reticular dermis. Furthermore, microscopic 1-D SHG polarimetry indicated a swell of the orientation angle and a large variance of the organization degree in the collagen fibers in the microscopic region. These results imply that the reticular dermis posses a tangled structure of collagen fibers, which is highly consistent with the result of the anatomical examination of the skin. The proposed method will be a powerful tool for monitoring the microscopic distribution of the collagen fiber orientation in the human dermis.

Optical coherence tomography (OCT) provides more parameters than pure morphology does. In a recent paper [A. Knuettel and M. Boehlau-Godau, J. Biomed. Opt. 5(1) 83–92 (2000)] we have shown that the refractive index (RI) can be evaluated in a localized manner in skin tissue under in vivo conditions. Based on a theory, originally developed for light detecting and ranging applications [L. Thrane et al., J. Opt. Soc. Am. A 17(3) 484–490 (2000)], the parameter mean scattering angle (MSA) could be derived in addition to RI. The effects of hydration on MSA and RI have been evaluated in vitro in pigskin and in vivo in human skin with our OCT scanner SkinDex 300®. These parameters may have a viable impact in (cosmetic) skin research and clinical diagnoses. To the best of our knowledge, this is the first time that (multiple) scattering of light has been quantified through the observation of a new scattering parameter under in vivo conditions.

Skin structure and age-related changes in human skin were characterized in vivo by applying confocal laser scanning microscopy (CLSM) and optical coherence tomography (OCT). The overall effect of aging skin, derived from studies of volunteers belonging to two age groups, was found to be a significant decrease in the maximum thickness of the epidermis and flattening of the dermo-epidermal junction. At a certain depth in the dermis, well below the basal layer, a reflecting layer of fibrous structure is observed in CLSM images. The location of this layer strongly depends on age and is situated much deeper below the skin surface in younger than in older skin. In addition, large structural changes were observed with age. The OCT images show two bright reflecting layers. The first one is due to scattering at the skin surface. The second band appears to be caused by a layer of fibrous structure in the dermis. Direct comparison of CLSM and OCT suggests that the same fibrous layer is imaged by the two techniques. This layer might be due to the transition between the papillary and reticular dermis. A comparison of CLSM and OCT enables a better understanding of the images.

The use of optical coherence tomography (OCT) for imaging skin during cryosurgery is evaluated. OCT provides high spatial resolution (5–10 µm) images of optical backscattering due to local variations in refractive index, such as the boundary between liquid and frozen water in tissue. Time resolved OCT images were acquired during freezing of water, Intralipid^TM, and in vivo hamster skin. Subsurface morphological changes were evident only during freezing of Intralipid and skin. A simple thermal model was applied which predicted freezing times on the same order of magnitude as those observed in OCT images. OCT can be used as a feedback tool during cryosurgical procedures to monitor progression of the freezing front.

Optical coherence tomography enables cross-sectional imaging of tissue structure to depths of around 1.5 mm, at high-resolution and in real time. Incorporation of polarization sensitivity (PS) provides an additional contrast mechanism which is complementary to images mapping backscattered intensity only. We present here polarization-sensitive optical coherence tomography (OCT) images of human skin in vivo, demonstrating the ability of the technique to visualize and quantify the birefringent properties of skin. Variation in normal skin birefringence according to anatomical location is demonstrated, and discussed in relation to collagen distribution at each location. From measurements on a sample of five human volunteers, mean double-pass phase retardation rates of 0.340±0.143, 0.250±0.076, and 0.592±0.142 deg/µm were obtained for the dorsal hand, temple, and lower back regions, respectively. We demonstrate how averaging the Stokes parameters of backscattered light over a range of axial and lateral dimensions results in a reduction of speckle-induced noise. Examples of PS-OCT images from skin sites following wound healing and repair are also presented and discussed.

Skin cancer is the most common human malignancy, with basal cell carcinoma (BCC) the most frequent type. Aggressive forms of BCC are associated with extensive dermal invasion and destruction of collagen. Surgery is the most common treatment, but identification of tumor borders is a challenge. Polarization-sensitive optical coherence tomography (PS-OCT) is an optical method to examine collagen birefringence. To date, it has not been exploited for cancer management. As part of a pilot exploratory study to examine the use of OCT in skin cancer, we examined several tumors that pose a challenge to the surgeon due to their large size and histological subtype. In normal perilesional skin, OCT identifies epidermal and dermal structure; PS-OCT identified dermal birefringence. In BCC, tumors lost normal structure and gained the appearance of lobular impressions. PS-OCT identified an alteration of dermal birefringence. Examination of a border area revealed a gradual transition from more normal appearing image to frank tumor. These results indicate that PS-OCT can identify features that distinguish normal skin from tumor and may have the potential to guide surgeons in the treatment of aggressive skin cancer.

We report on development of an optical-thermal model to evaluate the use of pulsed photothermal radiometry (PPTR) for depth profiling of port wine stain (PWS) skin. In the model, digitized histology sections of a PWS biopsy were used as the input skin geometry. Laser induced temperature profiles were reconstructed from simulated PPTR signals by applying an iterative, non-negatively constrained conjugate gradient algorithm. Accuracy of the following PWS skin characteristics extracted from the reconstructed profiles was determined: (1) average epidermal thickness (z_epi), (2) maximum epidermal temperature rise (ΔT_epi,max), (3) depth of PWS upper boundary (z_PWS), and (4) depth of maximum PWS temperature rise (z_PWS,max). Comparison of the actual and reconstructed profiles from PPTR data revealed a good match for all four PWS skin characteristics. Results of this study indicate that PPTR is a viable approach for depth profiling of PWS skin.

It is well known that the use of tightly focused continuous wave lasers can be an effective treatment of common telangiactasia. In general, the technique requires the skills of a highly dexterous surgeon using the aid of optical magnification. Due to the nature of this approach, it has proven to be largely impractical. To overcome this, we have developed an automated system that alleviates the strain on the user associated with the manual tracing method. The device makes use of high contrast illumination, simple monochromatic imaging, and machine vision to determine the location of blood vessels in the area of interest. The vessel coordinates are then used as input to a two-dimensional laser scanner via a near real-time feedback loop to target, track, and treat. Such mechanization should result in increased overall treatment success, and decreased patient morbidity. Additionally, this approach enables the use of laser systems that are considerably smaller than those currently used, and consequently the potential for significant cost savings. Here we present an overview of a proof-of-principle system, and results using examples involving in vivo imaging of human skin.

The chromatic characteristics of skin color arise from the interactions of light (primarily absorption and scattering) with the epidermis and the dermis. The primary light absorbers in skin are hemoglobin and melanin. Most of scattering is attributed to collagen fibers and in pigmented skin to melanosomes. Traditionally skin redness is considered to arise due to locally elevated concentrations of hemoglobin, whereas skin pigmentation is attributed to melanin. In this study we attempt to understand better the contributions of these chromophores to the perceived skin color using spectral analysis of skin color reactions induced by ultraviolet (UV) irradiation or pressure. In the first experiment 12 individuals with skin phototypes III–IV were irradiated on the back using a solar simulator with doses ranging from 0.7 to 3 MED. The skin reactions were evaluated on days 1, 7, 14, and 21 after irradiation. Evaluations included diffuse reflectance spectroscopy (DRS) and clinical assessment of the erythema and the pigment reaction. Apparent concentrations of melanin, oxy-, and deoxy-hemoglobin were calculated from the absorption spectra. In the second experiment the levels of deoxy-hemoglobin of the volar forearm of ten volunteers were selectively altered by either application of a pressure cuff or by topical application of 3% H₂O₂. Changes in skin color appearance were documented by photography, colorimetry, and DRS. In the UV exposure experiment all reactions were dose dependent. Oxy-hemoglobin values increased to a maximum on day 1, correlating well with the clinical evaluation of erythema, and then decreased exponentially to base line. Melanin showed a significant increase on day 7 and remained relatively constant for the next 3 weeks, correlating well with the clinical evaluation of pigmentation (tanning). Deoxy-hemoglobin increased slightly on day 1 and remained elevated for the next 2 weeks. Thus, deoxy-hemoglobin correlated moderately with the clinical erythema scoring on day 1 only, while it contributes significantly to what is clinically perceived as skin tanning on days 7 and 14. Application of pressure below the diastolic level increased deoxy-hemoglobin concentration as measured by DRS. This increase corresponded to a decrease of a "pigmentation" parameter (based on the L*a*b* scale) in a similar fashion that has been documented for increases in melanin concentration. Topical H₂O₂ application reduced deoxy-hemoglobin levels as measured by DRS. This reduction coincided kinetically with a visible skin blanching. Application of pressure or H₂O₂ did not significantly alter the levels of oxy-hemoglobin or melanin. In this report we present compelling evidence that deoxy-hemoglobin significantly contributes to the skin color appearance. Blood pooling, expressed as increased deoxy-hemoglobin, can contribute to what is visually perceived as pigmentation. Furthermore, we present that measurement of its contribution to the skin color appearance can only be accomplished with DRS.

Confocal reflectance microscopy of skin and other tissues in vivo is currently limited to imaging at the cellular, nuclear and general architectural levels due to the lack of microstructure-specific contrast. Morphologic and functional imaging at specific organelle and microstructure levels may require the use of exogenous contrast agents in small (nontoxic) concentrations, from which weakly backscattered light must be detected in real time. We report an analysis based on Mie theory to predict detectability, in terms of signal-to-background and signal-to-noise ratios, of reflectance contrast agents within skin and microcirculation. The analysis was experimentally verified by detectability of (a) intravenously injected polystyrene microspheres that enhance the contrast of dermal microcirculation in Sprague–Dawley rats, and (b) acetic acid-induced compaction of chromatin that enhances nuclear morphology in normal and cancerous human skin. Such analyses and experiments provide a quantitative basis for developing the opto-biochemical properties and use of contrast agents and for designing confocal instrumentation to enable real-time detectability in vivo.

The predominant long-chain carotenoids found in human skin are lycopene and β-carotene. They are powerful antioxidants and thought to act as scavengers for free radicals and singlet oxygen formed by normal metabolism as well as excessive exposure of skin to sunlight. The specific importance of the particular representatives of the carotenoid antioxidants regarding skin defense mechanisms is of strong current interest. We demonstrate fast and noninvasive detection of β-carotene and lycopene concentrations in living human skin using Raman detection of the molecules' carbon–carbon double bond stretch vibrations. Employing excitation with suitable blue and green laser lines, and taking advantage of differing Raman cross sectional profiles for β-carotene and lycopene, we determine the relative concentration of each carotenoid species. This novel technique permits the quantitative assessment of individual long-chain carotenoid species rather than their composite level in human skin. The obtained results reveal significant differences in the carotenoid composition of the subjects' skin and show that the ratio between β-carotene and lycopene concentration can vary from 0.5 to 1.6. The technique holds promise as a method for rapid screening of carotenoid compositions in human skin in large populations and should be suitable for clinical studies correlating carotenoid status with risk for cutaneous diseases.

Fluorescence diagnostic techniques are notable amongst many other optical methods because they offer high sensitivity and noninvasive measurement of tissue properties. However, a combination of multiple scattering and physical heterogeneity of biological tissues hampers interpretation of the fluorescence measurements. Analyses of the spatial distribution of endogenous and exogenous fluorophores excitation within tissues and their contribution to the detected signal localization are essential for many applications. We have developed a novel Monte Carlo technique that gives a graphical perception of how the excitation and fluorescence detected signal are localized in tissues. Our model takes into account the spatial distribution of fluorophores, the variation of concentrations and quantum yield. We demonstrate that matching the refractive indices of the ambient medium and topical skin layer improves spatial localization of the detected fluorescence signal within the tissues.

Modeling of skin burns has been performed in this study. Autocorrelation functions of intensity fluctuations of scattered light were measured for two-layered turbid media. The first layer served as a model for motionless scatterers (optically inhomogeneous gel film) whereas the second one simulated dynamic light scattering (Brownian motion of intralipid particles in aqueous suspension). This medium was used as a model of skin burns. A theory related quasi-elastic light scattering measurements to cutaneous blood flow was used. The dependencies of statistical properties of Doppler signal on the properties of burned skin as well as on the velocity of cutaneous blood flow have been investigated. Theoretical predictions have been verified by measurements both of dynamic and stationary light scattering in model media.

The physical basis for optimization of wavelength, pulse duration, and cooling for laser-induced selective photothermolysis of hair follicles in human skin is discussed. The results indicate that the most important optimization parameter is the cooling efficiency of the technique utilized for epidermal protection. The optical penetration is approximately the same for lasers at 694, 755, and 800 nm. The penetration of radiation from Nd:yttrium–aluminum–garnet lasers at 1064 nm is, however, somewhat larger. Photothermal damage to the follicle is shown to be almost independent of laser pulse duration up to 100 ms. The results reveal that epidermal cooling by a 30–80-ms-long cryogen spurt immediately before laser exposure is the only efficient technique for laser pulse durations less than 10 ms. For longer pulse durations in the 30–100 ms range, protection can be done efficiently by skin cooling during laser exposure. For laser pulses of 100 ms, an extended precooling period, e.g., by bringing a cold object into good thermal contact with the skin for about 1 s, can be of value. Thermal quenching of laser induced epidermal temperature rise after pulsed exposure can most efficiently be done with a 20 ms cryogen spurt applied immediately after irradiation.

In this paper, a new method on extraction of human skin grid centerlines is proposed. The method introduces the physics concepts of kinetic and potential energy into image processing. Regional energy is calculated. Energy transformation is performed to map the pixels from the grayscale space into energy space. Then, the energy image undergoes a morphological filter to remove noises and spurious minima. The amount of filtering can be manually tuned to get a different result. Subsequently, normal curvature of the energy surface is utilized to identify the principal direction and principal curvatures. The ridge centerlines can be detected at the image locations where the principal direction is perpendicular to the normal vector. The experiment shows that this method is an effective one for the purpose of extracting human skin grid.

Microsecond laser pulses are currently being investigated in a new ophthalmic procedure for treatment of disorders associated with the retinal pigment epithelium (RPE). The precise mechanism for microsecond laser-induced RPE damage, however, has not been determined. We have previously shown that short pulse laser irradiation in the nanosecond to picosecond time domain causes transient microbubble formation around melanin granules in pigmented cells. Nanosecond time-resolved microscopy was previously used to visualize the intracellular cavitation dynamics. However, this technique is difficult to use with microsecond laser exposures, especially when multiple laser pulses are applied in a rapid sequence as in the clinical setting. Here we describe a simple pump-probe method for detecting transient light scattering signal from individual RPE cells when they are irradiated with nanosecond and microsecond laser pulses. For single 12 ns pulses the threshold for bubble detection was the same as the ED₅₀ threshold for cell death. For 6 µs pulse duration the threshold for bubble detection was about 10% higher than the threshold for cell death. With repetitive pulse trains at 500 Hz the ED₅₀ decreased about 25% for 10 and 100 pulses. Cells die when a single bubble was detected in a multiple pulse sequence.

Confocal Raman piezo-spectroscopy has been used for the quantitative assessments of phase transformation and residual stresses in zirconia made artificial hip joints. This work can be considered to be a first step towards the development of a fully quantitative technique for the spectroscopic characterization of zirconia femoral heads and other zirconia parts for biomedical applications. After establishing reliable calibration procedures, Raman microprobe spectroscopy could be extended to provide quantitative assessments of zirconia metastability and microscopic stress fields along the z axis perpendicular to the joint surface. For the first time, we have directly visualized patterns of phase-transformation and related residual stresses on the very surface and along the subsurface of both in vitro tested and retrieved hip implants. These spectroscopic assessments may open a completely new perspective in understanding the micromechanical wear behavior of zirconia ceramics in biological environment and in developing new zirconia-based biomaterials with superior stability characteristics.

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.

This study investigates the feasibility of using the enhanced cyan mutant of green fluorescent protein (ECFP) as a probe for two-photon fluorescence correlation spectroscopy (FCS). Molecular dynamics and other properties of ECFP and an ECFP-tubulin fusion protein were investigated in living Potorous tridactylis (PTK2) cells. ECFP has high molecular brightness in the nucleus (η= 3.3 kcpsm) and in the cytoplasm (3.2 kcpsm) under our experimental conditions. The diffusion constants of ECFP were determined to be 20±7 µm²/s in the nucleus and 21±8 µm²/s in the cytoplasm. ECFP has stable molecular characteristics with negligible photobleaching and photodynamic effects in our measurements. At the highest concentration of monomer ECFP (425 nM) the amount of dimer ECFP was estimated to be negligible (~1.8 nM), consistent with our data analysis using a single species model. ECFP-tubulin has a diffusion constant of 6 µm²/s in the living cells. In addition, we demonstrate that analysis of the molecular brightness can provide a new avenue for studying the polymerization state of tubulin. We suggest that the tubulin in the vicinity of the nucleus exists primarily as a heterodimer subunit while those in the area away from the nucleus (d>5 µm) are mostly oligomers. We conclude that ECFP is a useful genetic fluorescent probe for FCS studies of various cellular processes when in fusion to other biomolecules of interest.

Infrared fingerprints of molecules in biology contain much information on cells metabolism allowing one to distinguish between healthy and altered tissues. Here, to collect infrared signatures, we used evanescent wave spectroscopy based on an original infrared transmitting tapered glass fiber. A strict control of the fiber diameter in the tapered sensing zone allows high sensitivity and wide spectral range exploration from 800 to 3000 cm^–1. Then, merely in depositing the mouse liver biopsies on the fiber, this device has enable us to differentiate between tumorous and healthy tissues.

Analysis of speckle dynamics is frequently used to study the motion of scattered objects or liquids. By assessing the increase in contrast on the speckle field produced by a blood plasma sample, illuminated by a laser during a coagulation test, as well as the slowing down of speckle fluctuations, we measured the time required for blood plasma coagulation in vitro and evidence the process dynamics. Then, we compared this noninvasive method with a mechanical viscosity-based detection system classically used in hematology laboratories; our results show good correlation and could provide more information about the blood clotting process.

Muscle blood flow (MBF) and muscle oxygen saturation (SmO₂) were measured at eight locations (four proximal, four distal) over a 4×8 cm² area of the vastus lateralis at rest and immediately after isometric, maximal quadriceps contraction using multichannel, frequency-domain, near-infrared spectroscopy. A venous occlusion was applied 20 s before the end of the exercise, so that the venous-occlusion-induced increase in total hemoglobin was recorded without any delay after the end of the exercise. Therefore, we were able to investigate the relationship between the exercise-induced changes in vastus lateralis MBF and SmO₂. After exercise, MBF increased significantly at each measured location. Comparing the MBF values measured at the end of exercise in the proximal and distal regions, we observed that only one proximal region had a significantly higher MBF than the corresponding distal one. The maximum desaturation measured during exercise was positively correlated with the postexercise to pre-exercise MBF ratio in both the proximal (P = 0.016) and distal (P = 0.0065) regions. These data confirm that frequency-domain tissue oximeters are noninvasive, powerful tools to investigate the spatial and temporal features of muscle blood flow and oxygenation, with potential applications in areas of pathophysiology.