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.     

SPECTROSCOPIC AND MICROSCOPIC CHARACTERISTICS OF HUMAN SKIN AUTOFLUORESCENCE EMISSION

To improve the understanding of human skin autofluorescence emission, the spectroscopic and microscopic characteristics of skin autofluorescence were studied using a combined fluorescence and reflectance spectroanalyzer and a fiber optic microspectrophotometer. The autofluorescence spectra of in vivo human skin were measured over a wide excitation wavelength range (350–470 nm). The excitation–emission matrices of in vivo skin were obtained. An excitation–emission maximum pair (380 nm, 470 nm) was identified. It was revealed that the most probable energy of skin autofluorescence emission photons increases monotonically and near linearly with increasing excitation photon energy. It was demonstrated that the diffuse reflectance, R, can be used as a first order approximation of the fluorescence distortion factor f to correct the measured in vivo autofluorescence spectra for the effect of tissue reabsorption and scattering. The microscopic in vitro autofluorescence properties of excised skin tissue sections were examined using 442 nm He–Cd laser light excitation as an example. It was demonstrated that the fluorophore distribution inside the skin tissue is not uniform and the shapes of the autofluorescence spectra of different anatomical skin layers vary. The result of this study confirms that the major skin fluorophores are located in the dermis and provides an excellent foundation for Monte Carlo modeling of in vivo autofluorescence measurements.     

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. High-quality in vivo NIR Raman spectra associated with an autofluorescence background from mouse skin and tumor tissue were acquired in 5 s. Multivariate statistical techniques, including principal component analysis (PCA) and linear discriminant analysis (LDA), were used to develop diagnostic algorithms for differentiating tumors from normal tissue based on their spectral features. Spectral classification of tumor tissue was tested using a leave-one-out, cross-validation method, and the receiver operating characteristic (ROC) curves were used to further evaluate the performance of diagnostic algorithms derived. Thirty-two in vivo Raman, NIR fluorescence and composite Raman and NIR fluorescence spectra were analyzed (16 normal, 16 tumors). Classification results obtained from cross-validation of the LDA model based on the three spectral data sets showed diagnostic sensitivities of 81.3%, 93.8% and 93.8%; specificities of 100%, 87.5% and 100%; and overall diagnostic accuracies of 90.6%, 90.6% and 96.9% respectively, for tumor identification. ROC curves showed that the most effective diagnostic algorithms were from the composite Raman and NIR autofluorescence techniques.     

The Dynamics of Laser-Induced Changes in Human Skin Autofluorescence—Experimental Measurements and Theoretical Modeling

To study the temporal dynamics of human skin autofluorescence photobleaching, we measured the autofluorescence spectral changes of skin in vivo during continuous exposure to 442 nm (He-Cd) laser light. Integral intensities were calculated for various spectral wavelength bands and plotted as a function of time. Mathematical analysis of the time function revealed a double-exponential photobleaching process: I(t) = a exp(-t/τ¹,) + b exp(-t/τ²) + c, in which t¹, and t² differed by an order of magnitude. A hypothesis for the mechanism of the double-exponential photobleaching dynamics was proposed and evaluated using Monte Carlo modeling of light propagation in the skin and autofluorescence escape from skin. By combining the fluorophore microdistributions, Monte Carlo simulation results and the variation in fluorescence decrease parameters (a, b, c, τ¹τ²) with increasing exposure intensities a biophysical explanation for the double-exponential photobleaching function was elucidated. The fast decrease term corresponds to laser-induced photobleaching in the stratum corneum, while the slow decrease term represents fluorophore changes in the dermis. The measured autofluorescence photobleaching dynamics can be used to determine the fractional contributions of different skin layers to the total autofluorescence signal measured in vivo.     

In vivo fluorescence spectroscopy of nonmelanoma skin cancer

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.     

Optical properties of human melanocytic nevi in vivo

We present an in vivo study of the optical properties of melanin present in melanocytic nevi of human subjects with Fitzpatrick skin type III (Caucasian descent) using optical spectroscopy. We show that the melanin absorption spectrum exhibits an exponential dependence on wavelength with a decay constant which follows a normal distribution characteristic of a random biological variable. Moreover, we demonstrate lack of correlation among melanin optical properties, melanin concentration and skin light scattering properties, which indicates that the true optical absorption of melanin can be measured free from confounding scattering effects. We also show that the average melanin absorption spectrum in vivo is in very good agreement with a previously reported oxygen photoconsumption action spectrum of melanin. Finally, we provide an overview of the emerging picture of the melanin absorption properties in vivo among various skin types and also among various skin lesions such as melanocytic nevi and melanoma.     

Influence of epidermal thickness,pigmentation and redness on skin autofluorescence

Detection of autofluorescence at the skin surface is highly influenced by melanin and hemoglobin. Epidermal absorption and scattering may also be an influencing factor and is represented in this article as a quantitative parameter, epidermal thickness. To examine this parameter we measured the 370 nm fluorescence in vivo after excitation with 330 nm and the 455 nm fluorescence after excitation with 330 and 370 nm. Measurements were performed on sun-exposed skin at the dorsal aspect of the forearm and shoulder and on nonexposed buttock skin. Skin pigmentation and redness of the same body sites were measured by reflectance spectroscopy. The thickness of the stratum corneum and the cellular part of epidermis was quantified by light microscopy of skin biopsies. Multiple regression analysis was used to find correlations between autofluorescence and the potential influencing factors. We found a highly significant correlation of skin autofluorescence with pigmentation and redness for both emission wavelengths (Em). A small but significant correlation to epidermal thickness was found only for excitation wavelength (Ex) 370 nm and Em455 nm if body site was included in the analysis. No correlation between Ex330:Em370 and Ex330:Em455 and thickness of epidermis was found. For practical use, correction of skin autofluorescence for pigmentation is essential, correction for redness is of less importance and correction for epidermal thickness is unnecessary.     

Accuracy of Noninvasive in vivo Measurements of Photosensitizer Uptake Based on a Diffusion Model of Reflectance Spectroscopy

Abstract— This study compares the photosensitizer concentration measured noninvasively in vivo by diffuse reflectance spectroscopy with the results of postmortem tissue solubilization and fluorometric assay. The reflectance spectrometer consists of a fiber optic surface probe, spectrometer and charge-coupled device (CCD) array detector. The surface probe has eight detection fibers separated from the light source fiber by distances ranging from 0.85 to 10 mm. The imaging spectrometer disperses the light from each detector fiber onto the two-dimensional CCD array, while maintaining spatial separation of each individual spectrum. A single exposure of the CCD therefore captures the reflectance spectrum at eight distances and over a range of 300 nm. From the spectra, the tissue's optical scattering and absorption coefficients are determined using a diffusion model of light propagation. Changes in the tissue absorption are used to estimate the photosensitizer concentration. Normal New Zealand White rabbits were injected with aluminum phthalocyanine tetrasulfonate (AlPcS4) and probe measurements made 24 h after injection on the dorsal skin, on muscle after surgically turning the skin back and on liver. For skin, the noninvasive estimate is proportional to the true concentration but low by a factor of 3. Based on Monte Carlo modeling of multilayered systems, this underestimate is attributed to the layered structure of the skin and nonuniform AIPcS4 distribution. A comparison of the noninvasive concentration estimates to the postmortem assay results finds good agreement for liver tissue even though application of the diffusion model is not strictly justified.     

Near-infrared Fluorescence Spectroscopy Detects Alzheimer's Disease In Vitro

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.     

Near-infrared dyes,nanomaterials and proteins

Near-infrared probes including dyes, nanoparticles and proteins have been particularly useful for in vivo imaging because of their ability to penetrate tissue deeper with minimal scattering and autofluorescence, opening the door to study cell biology in physiological conditions.     

Diagnostic potential of autofluorescence for an assisted intraoperative delineation of glioblastoma resection margins

The intrinsic autofluorescence properties of biological tissues can be affected by the occurrence of histological and biochemical alterations induced by pathological processes. In this study the potential of autofluorescence to distinguish tumor from normal tissues was investigated with the view of a real-time diagnostic application in neurosurgery to delineate glioblastoma resection margins. The autofluorescence properties of nonneoplastic and neoplastic tissues were analyzed on tissue sections and homogenates by means of a microspectrofluorometer, and directly on patients affected by glioblastoma multiforme, during surgery, with a fiber-optic probe. Scan-microspectrofluorometric analysis on tissue sections evidenced a reduction of emission intensity and a broadening of the main emission band, along with a redshift of the peak position, from peritumoral nonneoplastic to neoplastic tissues. Differences in both spectral shape and signal amplitude were found in patients when the glioblastoma lesion autofluorescence was compared with those of cortex and white matter taken as healthy tissues. Both biochemical composition and histological organization contribute to modify the autofluorescence emission of neoplastic, with respect to nonneoplastic, brain tissues. The differences found in the in vivo analysis confirm the prospects for improving the efficacy of tumor resection margin delineation in neurosurgery.     

Monte Carlo simulation of the backscatter region in photon-excited energy dispersive X-ray fluorescence spectra

The backscatter region of energy-dispersive X-ray fluorescence spectra obtained by a typical radioisotope system is examined in detail. A Monte Carlo simulation program which incorporates all the information on photon scattering processes including electron momentum distributions in target atoms, form factors and scattering factors for the cross-sections is presented. The program uses extensive variance reduction techniques and has the option of simulating any type of either single or any specified combination of multiple scatters or the complete process as appears in a real detector response spectrum. Based on several simulated spectra various conclusions are reached including that the energy spread of Compton peaks depends mainly on the broadening effect of electron momenta together with detection system resolution, and that the intensity of double scatters is roughly an order of magnitude lower than that of single scatters for thick targets.     

Reverse Monte Carlo modeling of the structure of colloidal aggregates

In this work we present results for the structure of aerogels coming from the diffusion-limited cluster aggregation simulation method. Pair distribution functions and structure factors, resulting from simulation, were considered as experimental input for reverse Monte Carlo modeling. The modeling yielded structural models with pair distribution functions and structure factors nearly identical to the results of the simulations. Particle configurations from both the simulations and reverse Monte Carlo modeling have been analyzed in terms of the distribution of the number of neighbors. It is suggested that the reverse Monte Carlo method, when applied to the structure factor, may be a suitable technique for the interpretation of experimental scattering data on colloidal aerogels.     

Optical properties of in vitro epidermis and their possible relationship with optical properties of in vivo skin.

Using a spectrophotometer with an internal integrating sphere, the absorption (mu a) and reduced scattering (mu s') coefficients of in vitro epidermis were evaluated from reflectance and transmittance measurements. mu a and mu s' varied from 24 to 0.2 cm-1, and from 32 to 21 cm-1 respectively, on passing from 400 to 800 nm. Moreover, using an external integrating sphere, the reflectance spectrum of in vivo skin was compared with the reflectance spectrum calculated with a Monte Carlo model, in which the mean values of mu a and mu s' and different anisotropy parameters were used as input data. In vivo results show that the principle of similarity is entirely valid for wavelengths greater than 600 nm and may be considered a good approximation in the 400-600 nm band, and suggest that optical characteristics of in vivo skin may be inferred from reflectance measurements.     

Calculation of the surface excitation parameter for Si and Ge from measured electron backscattered spectra by means of a Monte-Carlo simulation.

A new Monte Carlo method has been developed for simulating backscattered electron spectra, and this was applied for determining the surface excitation parameter (SEP). The simulation is based on direct tracking of electron trajectories in the solid, taking into account elastic and inelastic events. The elastic scattering cross sections are taken from literature, while inelastic cross section data are obtained by a fitting procedure. After some iterations, the program produces electron spectra fitting well to the experimental ones. Si and Ge electron spectra were simulated and SEP values were calculated. The SEP values are compared to other ones from literature.     

Three-dimensional modeling of EXAFS spectral mixtures by combining Monte Carlo simulations and target transformation factor analysis

We have developed a new method for the three-dimensional modeling of extended X-ray absorption fine structure (EXAFS) spectra which enables the extraction of the local structure of aqueous metal complexes from spectral mixtures of several components. The new method combines two techniques: Monte Carlo simulation and target transformation factor analysis (TFA). Monte Carlo simulation is used to create random arrangements between the X-ray absorbing metal ion and the ligand atoms, and to calculate the theoretical EXAFS spectrum of each arrangement. The theoretical EXAFS spectrum is then introduced as test spectrum in the TFA procedure, to test whether or not the test spectrum is likely to be a component of the spectral mixtures. This coupled procedure is repeated until the error in the test spectrum is minimized. The new method can thus be used to isolate and refine the structure of complexes from spectral mixtures and to determine their relative concentrations, solely on the basis of an estimate of a ligand structure. The performance of the proposed method is validated using uranium Liii-edge EXAFS spectra of binary mixtures of two uranium(VI) 3,4-dihydroxybenzoic acid complexes.     

FLUORESCENCE SPECTRA IN LUNG WITH PORPHYRIN INJECTION

The fluorescence emission spectra from human bronchial mucosa and tumors, before and after injection of dihematoporphyrin ether/ester, have been measured with an optical multichannel analyzer from 500 to 750 nm. Fluorescence was excited with a violet krypton ion laser (average wavelength 410 nm). The autofluorescence spectra decrease monotonically with increasing wavelength except for a small broad peak near 600 nm. The spectra from tumor sites, after injection of the fluorescent porphyrin, exhibit the characteristic fluorescence emission at 630 and 690 nm, added to the autofluorescence spectrum. The spectra from control or nontumor sites are similar but the magnitude of the component due to the injected porphyrin is smaller than at a tumor site. The magnitude ratio of tumor to control site fluorescence depends on concentration of the porphyrin, tumor thickness, and time after injection. Autofluorescence degrades contrast and thus makes very thin tumors difficult to image. Subtraction of the autofluorescence background is desirable.     

Fluorescence spectral imaging of dihydroxyacetone on skin in vivo

Dihydroxyacetone (DHA) has been proposed as a potential alternative to dansyl chloride for use as a fluorescence marker on skin to assess stratum corneum turnover time in vivo. However, the fluorescence from DHA on skin has not been adequately studied. To address this void, a noninvasive, noncontact spectral imaging system is used to characterize the fluorescence spectrum of DHA on skin in vivo and to determine the optimal wavelengths over which to collect the DHA signal that minimizes the contributions from skin autofluorescence. The DHA-skin fluorescence signal dominates the 580-680 nm region of the visible spectrum when excited with ultraviolet radiation in the 320-400 nm wavelength region (UVA). An explanation of the time-dependent spectral features is proposed in terms of DHA polymerization and binding to skin.     

Recent advances in the use of near-infrared quantum dots as optical probes for bioanalytical,imaging and solar cell application

Near-infrared quantum dots (NIR QDs) represent a powerful material and diagnostic tool owing to their long emission wavelength which extends into the near-infrared region where permeation depths are much larger and where the intrinsic absorbance and autofluorescence of tissue is much smaller compared to shortwave emitting QDs. We are reviewing here recent (2008–2013) methods for the preparation of NIR QDs, their (bio)chemical modifications, and their applications. The article is subdivided into the following sections: (a) Synthesis of NIR QDs; (b) modification of NIR QDs and probe preparation; (c) applications of NIR QDs (with subsections on fluorescence quenching and fluorescence enhancement-based bioanalytical detection, on fluorescence bioimaging, on uses in photovoltaic cells and solar cells, and on molecular detection based on electrogenerated chemiluminescence). We finally make conclusions and discuss current challenges, trends, and future applications. The review contains 119 references. Figure

This review systematically presents the development, preparation methods, modifications and bioapplications of Near-infrared quantum dots (NIR QDs). The review contains 126 references.     

Near-infrared dyes,nanomaterials and proteins

Taking the advantage of reduced scattering and low autofluorescence background, the NIR fluorescence probes, such as fluorescence proteins, organic molecules and nanoparticles, not only hold the promise of in vivo imaging of biological processes in physiology and pathology with high signal-to-noise ratio, but also for clinical diagnosis. In this review, we provide an overview of the recent progress on NIR probes, focusing on fundamental mechanisms of NIR dyes and nanoparticles, and protein engineering strategies for NIR proteins.