In vivo SPECTROPHOTOMETRIC EVALUATION OF NEOPLASTIC AND NON-NEOPLASTIC SKIN PIGMENTED LESIONS–I. REFLECTANCE MEASUREMENTS

Reflectance spectrophotometry from 400 to 800 nm on different cutaneous pigmented lesions, including primary and metastatic malignant melanoma, pigmented nevi, lentigo and seborrhoeic keratosis, has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of the different lesions manifest dissimilar patterns, particularly in the near IR region. Comparison of reflectance of nevi with that of malignant melanomas results in a highly significant difference (P less than 10(-6)) between the two samples. Though interpretation of the spectra remains difficult as a result of the complexity of the optical processes of scattering and absorption, our results suggest that a detailed analysis of the reflectance spectrum may give clinically useful information, and could be utilized as an aid in clinical diagnosis of cutaneous pigmented lesions, especially where malignant melanoma is concerned.     

In Vivo Optical Properties of Melanocytic Skin Lesions: Common Nevi, Dysplastic Nevi and Malignant Melanoma

We present an in vivo study of the optical properties of common nevi, dysplastic nevi and malignant melanoma skin lesions in human subjects. Reflectance spectra were measured on 1379 skin lesions, in the visible and near-infrared spectral regions, using a spectral imaging system, in a clinical setting. Analysis of the data using a reflectance model revealed differences between the optical properties of melanin present in nevi and melanoma lesions. These differences, which are in agreement with our previous observations on average reflectance spectra, may be potentially useful for the noninvasive characterization of pigmented skin lesions and the early diagnosis of melanoma.     

In vivo SPECTROPHOTOMETRIC EVALUATION OF NEOPLASTIC AND NON-NEOPLASTIC SKIN PIGMENTED LESIONS. II: DISCRIMINANT ANALYSIS BETWEEN NEVUS AND MELANOMA

Reflectance spectrophotometry from 420 to 780 nm on 31 primary melanoma and 31 benign nevi has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of melanoma and nevi manifest dissimilar patterns. From these spectra four variables, whose physical and/or physiological meanings remain to be investigated, have been derived. All of them are significantly different when compared between melanoma and nevi. A discriminant function between the two groups of lesions has been determined by using a stepwise discriminant analysis, resulting in a test with a sensitivity of 90.3% and a specificity of 77.4%. This method of discrimination between melanoma and nevi seems to have a discriminating power almost equal to that of a clinical judgement from a specialized medical doctor, thus suggesting a new method for screening skin pigmented lesions.     

Long-wavelength near-infrared spectroscopic imaging for in-vivo skin hydration measurements

A digital imaging system has been developed to collect skin hydration data. The system combines a near-infrared camera with a liquid-crystal tunable filter (LCTF) to acquire spectral images at multiple narrow wavelength bands between 960 and 1700 nm. Software has been developed to control the instrument and to process the data. Reflectance images were collected of subjects whose forearms had been treated to increase and decrease skin moisture. The infrared absorption band between 1400 and 1500 nm was used to calculate relative skin moisture, and the intensity of this band was plotted as a function of position in the form of a grayscale image. This is a rapid, non-contact and non-invasive technique to provide information on skin hydration of use to medical and cosmetic research and clinical practice.     

THE EVOLUTION OF PHOTODYNAMIC THERAPY TECHNIQUES IN THE TREATMENT OF INTRAOCULAR TUMORS

Abstract The techniques of photodynamic therapy (PDT) and the indications for its use in the treatment of intraocular tumors have evolved during the years in which it has been assessed in patients at our institution. It is now clear that transcorneal PDT delivered at a subthermal dose-rate to the surface of a pigmented lesion such as choroidal melanoma has little effect. In the absence of pigment, however, as in the case of retinoblastoma or amelanotic melanoma of the iris or choroid, the tumor kill attributed to PDT alone is significant. Data from animal tumor models in our institution and from patient studies elsewhere suggest that the addition of heat with the light delivery will predictably improve the outcome of the treatment of pigmented lesions. Ocular PDT delivered in conjunction with heat will be useful clinically as an adjunct to scleral plaque therapy by reducing the height of a lesion and concurrently the dose of radiation necessary at the base of the tumor for sterilization. Since the clinical tumoricidal effect of PDT is now known to be due at least in part to vascular damage, trans-scleral application of light to the base of melanomas and occlusion of its blood supply holds significant promise of efficacy with continued improvement of the light delivery system. Finally, a transpupillary approach to occlusion of the choroidal vascular supply to a melanoma by surrounding the tumor with photodynamic lesions may provide the best approach for ocular PDT as a primary therapy.     

Measuring surface topography with scanning electron microscopy. I. EZEImage: a program to obtain 3D surface data.

Scanning electron microscopy (SEM) is widely used in the science of materials and different parameters were developed to characterize the surface roughness. In a previous work, we studied the surface topography with fractal dimension at low scale and two parameters at high scale by using the variogram, that is, variance vs. step log-log graph, of a SEM image. Those studies were carried out with the FERImage program, previously developed by us. To verify the previously accepted hypothesis by working with only an image, it is indispensable to have reliable three-dimensional (3D) surface data. In this work, a new program (EZEImage) to characterize 3D surface topography in SEM has been developed. It uses fast cross correlation and dynamic programming to obtain reliable dense height maps in a few seconds which can be displayed as an image where each gray level represents a height value. This image can be used for the FERImage program or any other software to obtain surface topography characteristics. EZEImage also generates anaglyph images as well as characterizes 3D surface topography by means of a parameter set to describe amplitude properties and three functional indices for characterizing bearing and fluid properties.     

IMPROVING THE EFFICIENCY OF HEMATOPORPHYRIN PHOTOTHERAPY OF OCULAR TUMORS

Malignant melanoma of the choroid is the most common primary intraocular tumor in adult humans. Controversy exists over which is the most effective therapy. One therapeutic modality that has not been thoroughly investigated is hematoporphyrin derivative phototherapy (HPdPRT), a technique used successfully in the clinic on many non-ocular tumors.
The effect of HPd PRT on an ocular, amelanotic melanoma was evaluated using Greene melanoma implanted on pigmented rabbit iris. The parameters used clinically on non-ocular tumors (intravenous 2.5 mg HPd kg^-1 body weight and irradiation at 633 nm 48 h later) were totally ineffective in killing Greene melanoma implanted on the iris. The dose of dye (2.5–5 mg kg^-1), wavelength of light (500-700 nm), and illumination intensity were varied to determine the most efficient parameters for treating this tumor. The most important parameter was dye dose; increasing it to 5 mg kg^-1 resulted in some control of tumor growth. Administering 100% O₂ prior to and during irradiation also improved HPd PRT cytotoxicity. The use of pulsed light (pulses of 1 or 2 min) further enhanced killing and reduced the length of irradiation needed. These studies suggested that HPd PRT might be used efficiently on ocular melanomas.     

Monoline argon laser (514 nm) treatment of benign pigmented lesions with long pulse lengths.

A dichroic filter was adjusted in order to make use of the green line of an argon laser for the treatment of pigmented lesions (actinic and senile lentigo, ephelis, café-au-lait marks and spilus nevus). Using a power of 1.5 W, a spot size of 0.5 mm and pulse lengths of 200 or 300 ms, satisfactory elimination of 620 pigmented cutaneous lesions was achieved. Although the pulse lengths used were much longer than the thermal relaxation time of the melanosomes, the histology after treatment showed little damage of adjacent structures. Good restoration of the epidermis and a return of normal pigmentation led to excellent cosmetic results, with few complications.     

Multispectral optical imaging combined in situ with XPS or ToFSIMS and principal component analysis

All X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) instruments have optical cameras to image the specimen under analysis, and often to image the sample holder as it enters the system too. These cameras help the user find the appropriate points for analysis of specimens. However they seldom give as good images as stand‐alone bench optical microscopes, because of the limited geometry, source/analyser solid angle and ultra‐high‐vacuum (UHV) design compromises. This often means that the images displayed to the user necessarily have low contrast, low resolution and poor depth‐of‐field. To help identify the different regions of the samples present we have found it useful to perform multispectral imaging by illuminating the sample with narrow‐wavelength‐range light emitting diodes (LEDs). By taking an image under the illumination of these LEDs in turn, each at a successively longer wavelength, one can build up a set of registered images that contain more information than a simple Red–Green–Blue image under white‐light illumination. We show that this type of multispectral imaging is easy and inexpensive to fit to common XPS and ToF‐SIMS instruments, using LEDs that are widely available. In our system we typically use 14 LEDs including one emitting in the ultraviolet (so as to allow fluorescent imaging) and three in the near infra‐red. The design considerations of this system are discussed in detail, including the design of the drive and control electronics, and three practical examples are presented where this multispectral imaging was extremely useful. Copyright © 2016 The Authors Surface and Interface Analysis Published by John Wiley & Sons Ltd.     

Electrocatalytic oxidation of ethylene glycol: Part III. In-situ infrared reflectance spectroscopic study of the strongly bound species resulting from its chemisorption at a platinum electrode in aqueous medium

The adsorption of ethylene glycol (EG) at a platinum electrode in aqueous medium was investigated using Electrochemically Modulated Infrared Reflectance Spectroscopy.The adsorption appears to the dissociative at any pH. However, striking changes occur in the composition of the adsorbed layer when the pH is varied from acid to alkaline solutions. Thus, linearly bonded CO is dominant at pH ∼ 1, whereas almost equal amounts of bridge-bonded and linearly bonded CO species are found at pH ∼ 13.No other adsorbed residue has been detected in the experimental conditions observed and for the wavelength ranges investigated. But detailed analysis of the spectroscopic data leads to the conclusion that other adsorbed organic species might be present on the electrode surface, at least in acid medium.     

Epidemiological Support for an Hypothesis for Melanoma Induction Indicating a Role for UVA Radiation

An hypothesis for melanoma induction is presented: UV radiation absorbed by melanin in melanocytes generates products that may activate the carcinogenic process. Products formed by UV absorption in the upper layers of the epidermis cannot diffuse down as far as to the melanocytes. Thus, melanin in the upper layer of the skin may be protective, while that in melanocytes may be photocarcinogenic. Observations that support this hypothesis include: (1) Africans with dark skin have a reduced risk of getting all types of skin cancer as compared with Caucasians, but the ratio of their incidence rates of cutaneous malignant melanoma to that of squamous cell carcinoma is larger than the corresponding ratio for Caucasians. (2) Albino Africans, as compared with normally pigmented Africans, seem to have a relatively small risk of getting cutaneous malignant melanomas compared to nonmelanomas. This is probably also true for albino and normally pigmented Caucasians. (3) Among sun-sensitive, poorly tanning persons, frequent UV exposures are associated with increased risk of melanoma, whereas among sun-resistant, well-tanning persons, increased frequency of exposure is associated with decreased melanoma risk. (4) It is likely that UVA, being absorbed by melanin, might have a melanoma-inducing effect. This is in agreement with some epidemiological investigations which indicate that sun-screen lotions may not protect sufficiently against melanoma induction. The relative latitude gradient for UVA is much smaller than that for UVB. The same is true for the relative latitude gradient of cutaneous malignant melanoma as compared with squamous cell carcinoma and basal cell carcinoma. Under the assumption that the average slopes of the curves relating incidence rates with fluences of carcinogenic UV radiation are similar for melanomas and nonmelanomas, these facts are in agreement with the assumption that UVA plays a significant role in the induction of melanomas in humans. This is in agreement with the experimental results with Xiphophorus.     

煤燃烧过程中表面结构变化的SEM图像分析

介绍了SEM图像分析的多种方法,并讨论了各自的描述能力,利用这些方法分析煤在燃烧各阶段SEM图像,通过研究得到煤在燃烧过程中表面分形维数的变化规律,同时可获得表面孔的某些参数变化历程。分析表明,淮南低变质煤在850℃燃烧过程中,煤焦颗粒表面的分维数有一个先变低后变高的过程,煤焦表面分维数在2.2至2.5区间内变化;同时孔径分布由近双曲线分布变化为近正态分布,孔油加权平均形状因子增加,在研究过程中发现,不同的计算方法会对图像的分形维数值产生影响,确定合适的计算方法是分形理论在煤燃烧领域中应用的关键。通过比较发现Xie法较为适合计算煤焦表面SEM图像。     

Sensitivity of the retina to radiation damage as a function of wavelength.

Abstract—Exposure of the retina of the rhesus monkey to visible and infrared radiation from CW optical sources like the Sun, xenon lamps, etc. produces small lesions or scotomata which may be classified as thermal or photochemical, depending on the wavelength and duration of exposure. The action spectrum for the production of retinal lesions has been determined for eight monochromatic laser wavelengths extending from 1064 to 441 nm. The corneal power required to produce a lesion decreases by three orders of magnitude in going from 1064 to 441 nm. Exposure to 1064 nm radiation for 1000 s produces a typical thermal lesion at elevated retinal temperatures. whereas a 1000 s exposure to 441 nm light produces a photochemical lesion at power levels too low to raise the retinal temperature by an appreciable amount (<0.1°). The two types of lesion have entirely different characteristics as will be discussed in some detail. The photopathology of thc photochemical lesion has been studied at postexposure times ranging from 1 h to 90 days and will be demonstrated in a number of histological slides. Moreover, this photopathology correlates well with monocular visual acuity tests in the rhesus monkey as defined by the Landolt ring technique. To further elucidate the differential effects on the retina of short vs long wavelength CW radiation, we have divided a simulated solar spectrum at sea level into two spectral bands. 400–800 nm and 700–1400 nm, and determined the radiant exposures required to produce very mild lcsions on the rhesus retina for exposure times of 1, 10, 100 and 1000 s. To correlate our data with solar retinitis and eclipse blindness the image diameter or spot size on the retina was 159 μm, corresponding to the image size of the Sun on the human retina. Exposure to the 400–800 nm spectrum for durations of 10 s or greater required approximately 400 J/cm² to produce a mild photochemical lesion. Reciprocity is maintained over the exposure range 10–1000 s. Radiant exposure to the 700–1400 nm spectrum, on the other hand, required roughly 69,100 J/cm² for a 1000 s exposure. This was a mild thermal lesion. We were unable to produce a lesion for exposure times less than 1000 s. We interpret these data to mean that solar retinitis and eclipse blindness are primarily photochemical events produced by the short wavelength component of the solar spectrum, and that the infrared component of the solar spectrum plays only a minor role in these retinal pathologies.     

SENSITIVITY OF MOUSE SKH:HR-2 TO ULTRAVIOLET LIGHT: MOUSE PIGMENTATION MODEL

Abstract— The mouse Skh:HR-2 has been reported to be sensitized to ultraviolet light and become pigmented. Using three independent parameters associated with pigmentation. we have examined the ability of the mouse to become pigmented. The methods utilized were spectroscopic (skin color). histological (melanocyte density and epidermal thickness), and biochemical (tyrosinase activity). Following a two-week ultraviolet exposure, the mice were pigmented with the degree of pigment change related to the ultraviolet dose administered. Perturbations in skin color, epidermal thickness, melanocyte density, and tyrosinase activity were recorded. Mice were also examincd for their response to tyrosinc applied topically following each ultraviolet exposure. With the exccption of epidermal thickness. all the pigmentation parameters were accentuated when compared to results from ultraviolet exposure alone.     

DNA Damage, Repair and Photoadaptation in a Xiphophorus Fish Hybrid

Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F₁ hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.     

In vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancer. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals such as autofluorescence and scattering can decrease the image quality and, hence, the diagnosis potential of this imaging method. We have used an inexpensive dual excitation and/or emission wavelength approach in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. One excitation and/or emission wavelength is at the corresponding maximum of the fluorescent compound, while the second is at a nearby excitation/emission minimum. The first image contains the emission from the fluorescent compound used combined with the signal from the unwanted background. The second image provides an image of just the unwanted background signal. The difference of both images taken, thus gives a contrast enhanced image of the skin lesion. The method relies on the assumption that the background signal does not change significantly due to the small changes in wavelength for excitation or emission. Image ratio methods have already been applied towards diagnosis of basal cell carcinomas after administration of aminolevulinic acid-induced protoporphyin IX. In this study, we describe in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the image processing procedure.     

Photodynamic Effects of Hypericin on Lipid Peroxidation and Antioxidant Status in Melanoma Cells

Photodynamic-induced cytotoxicity by hypericin (HYP) was studied on three human melanoma cell lines: one pigmented cell line (G361) and two amelanotic cell lines (M18 and M6). No significant variation in the rate of uptake and in the maximum level of HYP incorporation for the different cells was observed. In the dark, no cytotoxicity was observed in the range0–10^?6M HYP for the three cell lines. Amelanotic cells were found to be more sensitive than pigmented cells to irradiation of HYP with visible light (Λ > 590 nm). In addition, for the three cell lines HYP-induced photocytotoxicity was found to be drug-dose and light-dose dependent. Under the conditions used, thiobarbituric acid-reacting substances (TBARs) were significantly increased in amelanotic cells after irradiation (P < 0.0001). By contrast, the amount of TBARS remained unchanged in pigmented cells. Antioxidant defenses including enzymes and glutathione (GSH) were assayed before and after HYP photosensitization. Significantly increased total SOD activity was observed after photosensitization for amelanotic cells (P < 0.05), while glutathione peroxidase (GSHPx) and catalase (Cat) activities but also GSH levels were significantly decreased (P < 0.01). In pigmented cells a significantly increased Cat activity was found (P < 0.05), whereas GSHPx was unaffected after irradiation. It can be inferred that (a) HYP may be an effective PDT agent for melanoma and (b) there is a relationship between melanin content and sensitivity to HYP phototoxicity in human melanoma cells.     

PHOTODYNAMIC THERAPY OF B16 PIGMENTED MELANOMA WITH LIPOSOME-DELIVERED Si(IV)-NAPHTHALOCYANINE

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (ɛ= 5 × 10⁵ M⁻¹ cm⁻¹), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg⁻¹ of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 μg g⁻¹, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm⁻²; at higher dose rates, thermal effects are likely to become important.     

Tomographic Spectral Imaging with Multivariate Statistical Analysis: Comprehensive 3D Microanalysis.

A comprehensive three-dimensional (3D) microanalysis procedure using a combined scanning electron microscope (SEM)/focused ion beam (FIB) system equipped with an energy-dispersive X-ray spectrometer (EDS) has been developed. The FIB system was used first to prepare a site-specific region for X-ray microanalysis followed by the acquisition of an electron-beam generated X-ray spectral image. A small section of material was then removed by the FIB, followed by the acquisition of another X-ray spectral image. This serial sectioning procedure was repeated 10-12 times to sample a volume of material. The series of two-spatial-dimension spectral images were then concatenated into a single data set consisting of a series of volume elements or voxels each with an entire X-ray spectrum. This four-dimensional (three real space and one spectral dimension) spectral image was then comprehensively analyzed with Sandia's automated X-ray spectral image analysis software. This technique was applied to a simple Cu-Ag eutectic and a more complicated localized corrosion study where the powerful site-specific comprehensive analysis capability of tomographic spectral imaging (TSI) combined with multivariate statistical analysis is demonstrated.     

Fourier Transform Multipixel Spectroscopy and Spectral Imaging of Protoporphyrin in Single Melanoma Cells

Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo-PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge-coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (10³-⁻4 × 10³ point pixels in a single cell). The net result was the intensity I (x, y, λ), for each pixel of the image (x, y), at any wavelength (λ). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5-aminolevulinic acid (ALA). The fluorescence image of ALA-treated cells revealed endo-PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one sub-cellular site to another. Photoirradiation of the cells induced point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral-image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal-lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo-products could be visualized at sites of formation in suhcellular compartments.