Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

BACKGROUND: Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. METHODS: We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 µm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. RESULTS: MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. CONCLUSION: This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ.

Label-free identification and characterization of murine hair follicle stem cells located in thin tissue sections with Raman micro-spectroscopy.

Stem cells offer tremendous opportunities for regenerative medicine. Over the past decade considerable research has taken place to identify and characterize the differentiation states of stem cells in culture. Raman micro-spectroscopy has emerged as an ideal technology since it is fast, nondestructive, and does not require potentially toxic dyes. Raman spectroscopy systems can also be incorporated into confocal microscope imaging systems allowing spectra to be obtained from below the tissue surface. Thus there is significant potential for monitoring stem cells in living tissue. Stem cells that reside in hair follicles are suitable for testing this possibility since they are close to the skin surface, and typically clustered around the bulge area. One of the first steps needed would be to obtain Raman micro-spectra from stem cells located in thin sections of tissue, and then see whether these spectra are clearly different from those of the surrounding differentiated cells. To facilitate this test, standard 5 µm thick sections of murine skin tissue were stained to identify the location of hair follicle stem cells and their progeny. Raman spectra were then obtained from adjacent cells in a subsequent unstained 10 µm thick section. The spectra revealed significant differences in peak intensities associated with nucleic acids, proteins, lipids and amino acids. Statistical analyses of the Raman micro-spectra identified stem cells with 98% sensitivity and 94% specificity, as compared with a CD34 immunostaining gold standard. Furthermore analyses of the spectral variance indicated differences in cellular dynamics between the two cell groups. This study shows that Raman micro-spectroscopy has a potential role in identifying adult follicle stem cells, laying the groundwork for future applications of hair follicle stem cells and other somatic stem cells in situ.

Imaging directed photothermolysis through two-photon absorption demonstrated on mouse skin - a potential novel tool for highly targeted skin treatment.

One-photon absorption based traditional laser treatment may not necessarily be selective at the microscopic level, thus could result in un-intended tissue damage. Our objective is to test whether two-photon absorption (TPA) could provide highly targeted tissue alteration of specific region of interest without damaging surrounding tissues. TPA based laser treatments (785 nm, 140 fs pulse width, 90 MHz) were performed on ex vivo mouse skin using different average power levels and irradiation times. Reflectance confocal microscopy (RCM) and combined second-harmonic-generation (SHG) and two-photon fluorescence (TPF) imaging channels were used to image before, during, and after each laser treatment. The skin was fixed, sectioned and H & E stained after each experiment for histological assessment of tissue alterations and for comparison with the non-invasive imaging assessments. Localized destruction of dermal fibers was observed without discernible epidermal damage on both RCM and SHG + TPF images for all the experiments. RCM and SHG + TPF images correlated well with conventional histological examination. This work demonstrated that TPA-based light treatment provides highly localized intradermal tissue alteration. With further studies on optimizing laser treatment parameters, this two-photon absorption photothermolysis method could potentially be applied in clinical dermatology.

Durable rough skin phantoms for optical modeling.

Skin phantoms are often used to study and model light propagation. However, existing skin phantoms overlook the important effect of surface roughness on light propagation patterns. This paper reports the construction of durable phantoms with controllable surface roughness and bulk optical properties. With silica microspheres as the scattering particles, we theoretically model the scatterer density required to achieve the desired phantom optical properties before fabrication. The surface roughness and the attenuation coefficients of the constructed phantoms were validated using optical profilometry and ballistic spatial filter photometry. These rough skin phantoms were originally developed for laser speckle studies, but could also be used for studying optical phenomena where light experiences surface and bulk scattering at the same time.

A method for accurate in vivo micro-Raman spectroscopic measurements under guidance of advanced microscopy imaging.

The movement from the subjects during in vivo confocal Raman spectral measurements could change the measurement volume, leading to non-specific signals and inaccurate interpretation of the acquired spectrum. Here we introduce a generally applicable method that includes (1) developing a multimodal system to achieve real-time monitoring of every spectral measurement with reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) imaging; (2) performing region-of-interest measurement by scanning an area of the tissue during spectral acquisition. The developed method has been validated by measuring different micro-structures of in vivo human skin. Our results demonstrated great consistency between RCM images and confocal Raman spectra. The superior quality of the images and spectra allows us to derive blood flow velocity and blood glucose level. We believe this method is valuable for realizing accurate microscopic spectral measurement and have great potential to be adapted into clinic to achieve non-invasive measurement of important biological parameters.

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin.

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two-photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique.

Polarization speckle imaging as a potential technique for in vivo skin cancer detection.

Skin cancer is the most common cancer in the Western world. In order to accurately detect the disease, especially malignant melanoma-the most fatal form of skin cancer-at an early stage when the prognosis is excellent, there is an urgent need to develop noninvasive early detection methods. We believe that polarization speckle patterns, defined as a spatial distribution of depolarization ratio of traditional speckle patterns, can be an important tool for skin cancer detection. To demonstrate our technique, we conduct a large in vivo clinical study of 214 skin lesions, and show that statistical moments of the polarization speckle pattern could differentiate different types of skin lesions, including three common types of skin cancers, malignant melanoma, squamous cell carcinoma, basal cell carcinoma, and two benign lesions, melanocytic nevus and seborrheic keratoses. In particular, the fourth order moment achieves better or similar sensitivity and specificity than many well-known and accepted optical techniques used to differentiate melanoma and seborrheic keratosis.

Differentiation of HaCaT cell and melanocyte from their malignant counterparts using micro-Raman spectroscopy guided by confocal imaging.

BACKGROUND: Skin cancer is the most common type of cancer in humans. Current techniques for identifying normal and neoplastic tissues are either destructive or not sensitive and specific enough. Raman spectroscopy and confocal imaging may obviate many limitations of existing methods by providing noninvasive, high-resolution, and real-time morphological and biochemical analysis of living tissues and cells. METHODS: We conducted micro-Raman spectroscopy studies on HaCaT cells, melanocytes (MC) and their malignant counterparts squamous cell carcinoma (SCC) and melanoma (MM) cells, respectively. Reflectance confocal imaging is used as guidance for the spectral measurements. RESULTS: Significant differences were found between the spectra of HaCaT cells and SCC cells, MC cells and MM cells, as well as all normal cells (HaCaT and MC) and all tumor cells (SCC and MM). Approximately 90% sensitivity and specificity was achieved for all the separations that we performed. CONCLUSION: Our results demonstrated the robust capability of confocal Raman spectroscopy in separating different cell lines. The acquired Raman spectra of major types of skin cells and their malignant counterparts will be useful for the interpretation of Raman spectra from in vivo skin. We believe it will eventually help diagnosis of skin cancer and other skin disease in clinical dermatology.

40-year trends in skin cancer in British Columbia, Canada, 1973 to 2003.

BACKGROUND: Skin cancer is common in North America. Incidence rate trends are potentially important in the assessment of the effects of measures to increase sun awareness in the population as well as measures to reduce sun damage. OBJECTIVE: To determine the incidence of basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and cutaneous malignant melanoma (CMM) in a geographically defined Canadian population over a 40-year period. METHODS: Data were obtained from the BC Cancer Registry for the calendar years 1973, 1983, 1993, and 2003. RESULTS: Age-standardized incidence rates increased significantly from 1973 to 2003 for BCC, SCC, and CMM. LIMITATIONS: The ethnic makeup of British Columbia has changed over time, and a novel method of accounting for the effect of this on skin cancer rates is presented. CONCLUSION: The incidence rate for skin cancers continued to rise in British Columbia, but there appears to have been a decline in the incidence of CMM and BCC in the youngest cohorts.

Conditional random fields and supervised learning in automated skin lesion diagnosis.

Many subproblems in automated skin lesion diagnosis (ASLD) can be unified under a single generalization of assigning a label, from an predefined set, to each pixel in an image. We first formalize this generalization and then present two probabilistic models capable of solving it. The first model is based on independent pixel labeling using maximum a-posteriori (MAP) estimation. The second model is based on conditional random fields (CRFs), where dependencies between pixels are defined using a graph structure. Furthermore, we demonstrate how supervised learning and an appropriate training set can be used to automatically determine all model parameters. We evaluate both models' ability to segment a challenging dataset consisting of 116 images and compare our results to 5 previously published methods.

In vivo video rate multiphoton microscopy imaging of human skin.

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.

Generalizing common tasks in automated skin lesion diagnosis.

We present a general model using supervised learning and MAP estimation that is capable of performing many common tasks in automated skin lesion diagnosis. We apply our model to segment skin lesions, detect occluding hair, and identify the dermoscopic structure pigment network. Quantitative results are presented for segmentation and hair detection and are competitive when compared to other specialized methods. Additionally, we leverage the probabilistic nature of the model to produce receiver operating characteristic curves, show compelling visualizations of pigment networks, and provide confidence intervals on segmentations.

Spectroscopic characterization and microscopic imaging of extracted and in situ cutaneous collagen and elastic tissue components under two-photon excitation.

BACKGROUND/PURPOSES: Understanding the two-photon excitation spectral characteristics and microscopic morphology of cutaneous collagen and elastic tissue components is important for applying multiphoton microscopy (MPM) in basic skin biology research and for clinical diagnosis. METHODS: We developed a system for two-photon excitation spectral measurements at various excitation wavelengths. The microscopic morphology was studied using a commercial multiphoton microscope. RESULTS: We obtained two-photon excitation fluorescence (TPEF) excitation-emission matrices (EEM), for the first time, of purified collagen and elastin samples, as well as in situ collagen and elastic fibers within excised human dermis. The EEM of the dermis was found to be similar to that of elastin. The excitation spectra for second harmonic generation (SHG) from purified collagen and excised dermis were also studied and were found to have similar spectral shapes. CONCLUSION: This study, using the EEM spectroscopic approach, confirmed a previous imaging study inference that in the dermis, TPEF predominantly originates from elastic fibers, while SHG originates solely from collagen fibers. The EEM data and SHG excitation spectra obtained in this study can be used to guide the selection of excitation wavelengths for MPM applications in basic skin biology research and for clinical diagnosis.

Near-infrared autofluorescence imaging of cutaneous melanins and human skin in vivo.

In recent years, near-infrared (NIR) autofluorescence imaging has been explored as a novel technique for tissue evaluation and diagnosis. We present an NIR fluorescence imaging system optimized for the dermatologic clinical setting, with particular utility for the direct characterization of cutaneous melanins in vivo. A 785-nm diode laser is coupled into a ring light guide to uniformly illuminate the skin. A bandpass filter is used to purify the laser light for fluorescence excitation, while a long-pass filter is used to block the main laser wavelength but pass the spontaneous components for NIR reflectance imaging. A computer-controlled filter holder is used to switch these two filters to select between reflectance and fluorescence imaging modes. Both the reflectance and fluorescence photons are collected by an NIR-sensitive charge-coupled device (CCD) camera to form the respective images. Preliminary results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence than surrounding normal tissue. This confirmed our previous findings from NIR fluorescence spectroscopy study of cutaneous melanins and provides a new approach to directly image the distributions of cutaneous melanins in the skin. In-vivo NIR autofluorescence images may be useful for clinical evaluation and diagnosis of pigmented skin lesions, including melanoma.

Integrated real-time Raman system for clinical in vivo skin analysis.

BACKGROUND: Raman spectroscopy is a non-invasive optical technique that can probe the molecular structure and conformation of biochemical constituents. The probability of Raman scattering is exceedingly low ( approximately 10(-10)), and consequently up to now the practical application of Raman spectroscopy to clinical medicine has been limited by either the weak spectral signal or by the long data acquisition times. Recent advances in Raman hardware and probe design have reduced spectral acquisition times, paving the way for clinical applications. METHODS: We present an integrated real-time Raman spectroscopy system for skin evaluation and characterization, which combines customized hardware features and software implementation. Real-time data acquisition and processing includes CCD dark-noise subtraction, wavelength calibration, spectral response calibration, intensity calibration, signal saturation detection, cosmic ray rejection, fluorescence background removal, and composition modeling. Real-time in vivo Raman measurement of volar forearm skin is presented to illustrate the methods and modeling. RESULTS: The system design implemented full-chip vertical hardware binning to improve the signal-to-noise ratio by 16-fold. The total time for a single in vivo measurement with analysis can be reduced to 100 ms with this implementation. Human skin was well modeled using the base Raman spectra. CONCLUSION: In vivo real-time Raman can be a very promising research and practical technique for skin evaluation.

Real-time Raman spectroscopy for non-invasive skin cancer detection - preliminary results.

Raman spectroscopy is a non-invasive optical technique, which can assess molecular structures and conformations within biological tissue. The probability of Raman scattering is inherently low such that previous clinical applications of Raman spectroscopy have been limited by long data acquisition times. We have developed a rapid real-time Raman spectrometer system with measurement times of less than 1 second, paving the way for clinical application. In this presentation, we report preliminary clinical results for this real-time Raman system. To date 289 skin cancers and benign skin lesions have been measured. Using partial least squares regression and linear discriminant analysis to analyze the Raman spectra we found that skin cancers could be well differentiated from benign skin lesions (sensitivity 91% and specificity 75%) and malignant melanoma from benign pigmented lesions (sensitivity 97%, specificity 78%).

Automated autofluorescence background subtraction algorithm for biomedical Raman spectroscopy.

A significant advantage of Raman spectroscopy as a noninvasive optical technique is its ability to detect subtle molecular or biochemical signatures within tissue. One of the major challenges for biomedical Raman spectroscopy is the removal of intrinsic autofluorescence background signals, which are usually a few orders of magnitude stronger than those arising from Raman scattering. A number of methods have been proposed for fluorescence background removal including excitation wavelength shifting, Fourier transformation, time gating, and simple or modified polynomial fitting. The single polynomial and the modified multi-polynomial fitting methods are relatively simple and effective, and thus are widely used in biological applications. However, their performance in real-time in vivo applications and low signal-to-noise ratio environments is sub-optimal. An improved automated algorithm for fluorescence removal has been developed based on modified multi-polynomial fitting, but with the addition of (1) a peak-removal procedure during the first iteration, and (2) a statistical method to account for signal noise effects. Experimental results demonstrate that this approach improves the automated rejection of the fluorescence background during real-time Raman spectroscopy and for in vivo measurements characterized by low signal-to-noise ratios.

Toward instrument-independent quantitative measurement of fluorescence intensity in fiber-optic spectrometer systems.

Fluorescence has been widely used in biological research and clinical diagnosis. One challenge facing the rapid growth of fluorescence application is the inability to make comparable fluorescence intensity measurements during a long period of clinical study and across laboratories. We propose a method to implement system-independent fluorescence intensity calibration in fiber-optic fluorescence spectrometer systems. This method is based on a National Institute for Standards and Technology traceable standard light source for system spectral response calibration, and a fluorescence reference standard for fluorescence intensity calibration. Human skin in vivo and a fluorescence phantom made of fluorescent microspheres were measured with two different system configurations and at different probe-sample distances. Experimental results showed very good agreement with theory after system-independent fluorescence intensity calibration.

Monte Carlo simulation of cutaneous reflectance and fluorescence measurements--the effect of melanin contents and localization.

Melanin content and distribution in skin were studied by examining a patient with white, brown and blue skin tones expressed on skin affected by vitiligo. Both diffuse reflectance and autofluorescence spectra of the three distinction skin sites were measured and compared. Monte Carlo simulations were then performed to help explain the measured spectral differences. The modeling is based on a six-layer skin optical model established from published skin optical parameters and by adding melanin content into different locations in the model skin. Both the reflectance and fluorescence spectra calculated by Monte Carlo (MC) simulation were approximately in agreement with experimental results. The study suggests that: (1) trichrome vitiligo skin may be an ideal in vivo model for studying the effect of skin melanin content and distribution on skin spectroscopy properties. (2) Based on the skin optical model and MC simulation, the content and distribution of melanin in skin, or other component of skin could be simulated and predicted. (3) Both reflectance and fluorescence spectra provided information about superficial skin structures but fluorescence spectra are capable of providing information from deeper cutaneous structures. (4) The research method, including the spectral ratio method, the method of adding and modifying the melanin content in skin optical models, and MC simulation could be applied in other non-invasive optical studies of the skin.

Cutaneous melanin exhibiting fluorescence emission under near-infrared light excitation.

Under ultraviolet and visible light excitation, melanin is essentially a nonfluorescent substance. This work reports our study on near-infrared (NIR) fluorescence properties of melanins, and explores potential applications of NIR fluorescence techniques for evaluating skin disorders involving melanin. The NIR fluorescence spectrum is obtained using a fiber optic NIR spectrometer under 785-nm laser excitation. In vitro measurements are performed on synthetic dihydroxyphenylalanine (DOPA) melanin, melanin extracted from Sepia ink sacs, human hair, animal fur, and bird feathers. Paired spectral comparisons of white and black skin appendages show that melanization of hair, fur, or feathers more than doubles the NIR fluorescence. In vivo NIR autofluorescence of normal dorsal and volar forearm skin of 52 volunteers is measured. Dorsal forearm skin, which is darker than volar skin, exhibits significantly greater NIR fluorescence. Patients with vitiligo (n=4), compound nevus (n=3), nevus of Ota (n=1), superficial spreading melanoma (n=3), and postinflammatory hyperpigmentation (n=1) are also evaluated. NIR fluorescence is greater within the lesion than the surrounding normal skin for all these conditions except vitiligo, where the converse was true. The observed melanin NIR fluorescence provides a new approach to in vitro and in vivo melanin detection and quantification that may be particularly useful for evaluating pigmented skin lesions.