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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Counting moles automatically from back images.

Density of moles is a strong predictor of malignant melanoma, therefore, enumeration of moles is often an integral part of many studies that look at malignant melanoma. An automatic method of segmenting and counting moles would help standardize studies, compared with manual counting. We have developed an unsupervised algorithm for segmenting and counting moles from two-dimensional color images of the back torso region, as part of a study to evaluate the effectiveness of sunscreen.

Raman spectroscopy in combination with background near-infrared autofluorescence enhances the in vivo assessment of malignant tissues.

The diagnostic ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy, NIR autofluorescence spectroscopy and the composite Raman and NIR autofluorescence spectroscopy, for in vivo detection of malignant tumors was evaluated in this study. A murine tumor model, in which BALB/c mice were implanted with Meth-A fibrosarcoma cells into the subcutaneous region of the lower back, was used for this purpose. A rapid-acquisition dispersive-type NIR Raman system was employed for tissue Raman and NIR autofluorescence spectroscopic measurements at 785-nm laser excitation.

Raman spectroscopy of in vivo cutaneous melanin.

We successfully acquire the in vivo Raman spectrum of melanin from human skin using a rapid near-infrared (NIR) Raman spectrometer. The Raman signals of in vivo cutaneous melanin are similar to those observed from natural and synthetic eumelanins. The melanin Raman spectrum is dominated by two intense and broad peaks at about 1580 and 1380 cm(-1), which can be interpreted as originating from the in-plane stretching of the aromatic rings and the linear stretching of the C-C bonds within the rings, along with some contributions from the C-H vibrations in the methyl and methylene groups.

Laser-induced autofluorescence microscopy of normal and tumor human colonic tissue.

Laser-induced autofluorescence (LIAF) spectroscopy has been found to be a promising tool for early cancer diagnosis in various organs, but the reasons responsible for the spectral differences between normal and diseased tissue are still not well understood. In this study, a microspectrophotometer (MSP) system was used to identify the microscopic origins of tissue autofluorescence in the colon under the excitation of a helium-cadmium laser at 442 nm. Colonic tissue samples (normal: n=8, adenocarcinoma: n=10) were obtained from 12 patients with known or suspected malignancies of the colon.

Restoration of hair growth with topical diphencyprone in mouse and rat models of alopecia areata.

Background: The contact sensitizer, diphencyprone (DPCP), is one of the most effective therapies for the more severe forms of alopecia areata (AA).

Objective: The purpose of this study was to determine the efficacy of topical DPCP on the 2 available rodent models for AA, and to determine the underlying therapeutic mechanisms.

Methods: AA-affected mice and rats were treated unilaterally with topical DPCP on the ventral and dorsal surface, respectively.