Optical sensing for tumor detection in the liver.

BACKGROUND: There is an increasing trend for optical guidance techniques in surgery. Optical imaging using Diffuse Reflectance Spectroscopy (DRS) can distinguish different tissue types through a specific "optical fingerprint". We investigated whether DRS could discriminate metastatic tumor tissue from normal liver tissue and thus if this technique would have potential for further implementation into surgical instruments or radiological intervention tools. METHODS: A miniaturized optical needle was developed able to collect DRS spectra between 500 and 1600 nm. Liver specimen of 24 patients operated for colorectal liver metastases were analyzed with DRS immediately after resection. Multiple measurements were performed and DRS results were compared to the histology analysis of the measurement locations. In addition, normal liver tissue was scored for the presence or absence of steatosis. RESULTS: A total of 780 out of the 828 optical measurements were correctly classified into either normal or tumor tissue. The resulting sensitivity and specificity were both 94%. The results of the analysis for each patient individually showed an accuracy of 100%. The Spearman's rank correlation of DRS-estimated percentages of hepatic steatosis in liver tissue compared to that of the pathologist was 0.86. CONCLUSIONS: DRS demonstrates a high accuracy in discriminating normal liver tissue from colorectal liver metastases. DRS can also predict the degree of hepatic steatosis with high accuracy. The technique, here demonstrated in a needle like probe, may as such be incorporated into surgical tools for optical guided surgery or percutaneous needles for radiological interventions.

Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin.

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.

In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.

Confocal Raman spectroscopy is introduced as a noninvasive in vivo optical method to measure molecular concentration profiles in the skin. It is shown how it can be applied to determine the water concentration in the stratum corneum as a function of distance to the skin surface, with a depth resolution of 5 microm. The resulting in vivo concentration profiles are in qualitative and quantitative agreement with published data, obtained by in vitro X-ray microanalysis of skin samples. Semi-quantitative concentration profiles were determined for the major constituents of natural moisturizing factor (serine, glycine, pyrrolidone-5-carboxylic acid, arginine, ornithine, citrulline, alanine, histidine, urocanic acid) and for the sweat constituents lactate and urea. A detailed description is given of the signal analysis methodology that enables the extraction of this information from the skin Raman spectra. No other noninvasive in vivo method exists that enables an analysis of skin molecular composition as a function of distance to the skin surface with similar detail and spatial resolution. Therefore, it may be expected that in vivo confocal Raman spectroscopy will find many applications in basic and applied dermatologic research.

Simulation of color of port wine stain skin and its dependence on skin variables.

BACKGROUND AND OBJECTIVE: The understanding of why Port Wine Stain (PWS) skin is redder and darker as compared to normal skin has so far been based on qualitative analysis. This study aims at quantitatively analyzing the influence of skin anatomy variables on perceived skin color. STUDY DESIGN/MATERIALS AND METHODS: Reflectance spectra for visible light from normal and Port Wine Stain skin have been calculated using a Monte Carlo algorithm applied to a multi-layered skin model. Skin parameters that were varied are pigmentation, dermal scattering, dermal blood concentration, blood oxygenation, vessel diameter, and vessel depth. The CIE 1976 color system was used to interpret the resulting spectra as colors. RESULTS: A reduced dermal blood content results in a less red and lighter color. Distribution of a constant volume of blood in smaller vessels results in a redder and darker color. Skin with higher dermal scattering was calculated to be yellower and lighter and skin with increased epidermal pigmentation results in a yellower and darker color. CONCLUSIONS: Redness of PWS skin depends on both the concentration of dermal blood as well as on how it is distributed.

In vitro and in vivo Raman spectroscopy of human skin.

Noninvasive techniques that provide detailed information about molecular composition, structure, and interactions are crucial to further our understanding of the relation between skin disease and biochemical changes in the skin, as well as for the development of penetration enhancers for transdermal drug administration. In this study we present in vitro and in vivo Raman spectra of human skin. Using a Raman microspectrometer, in vitro spectra were obtained of thin cross sections of human skin. They provided insight into the molecular composition of different skin layers. Evidence was found for the existence of a large variation in lipid content of the stratum corneum. A simple experimental setup for in vivo confocal Raman microspectroscopy of the skin was developed. In vivo Raman spectra of the stratum corneum were obtained at different positions of the arm and hand of three volunteers. They provided evidence for differences in the concentration of natural moisturizing factor at these positions.

Optical absorption of blood depends on temperature during a 0.5 ms laser pulse at 586 nm.

Optical properties are important parameters in port wine stain laser treatment models. In this study we investigated whether changes in blood optical properties occur during a 0.5 ms laser pulse. Blood from three volunteers was irradiated in vitro with laser pulses (radiant exposure 2-12 J cm-2, wavelength 586 nm, pulse length 0.5 ms). Reflection and transmission coefficients, measured using double integrating spheres, decreased slightly during the first part of the pulse. At 2.9 J cm-2 radiant exposure, the reflectance increased, independent of total radiant exposure of the pulse. This was caused by blood coagulation. A second sudden increase in reflection and a significant increase in transmission occurred near 6.3 J cm-2 and was accompanied by a "popping" sound, indicating rapid expansion of bubbles due to blood vaporization. A multilayered model of blood was used to fit calculated transmission coefficient curves to the measurements and determine temperature-dependent optical blood absorption. Heat diffusion was shown to be of minor importance. A 2.5-fold increase in absorption for temperatures increasing from 20 to 100 degrees C, accurately describes transmission coefficients measured up to 2.9 J cm-2.

Band analysis of hydrated human skin stratum corneum attenuated total reflectance fourier transform infrared spectra in vivo.

Water content is an important factor for skin condition. The determination of the hydration state of the skin is necessary to obtain basic knowledge about the penetration and loss of water in the skin stratum corneum. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is used to measure hydration of the stratum corneum. In this study we apply direct band fitting of the water bending, combination, and OH stretch bands over the 4000-650 cm-1 wave number range. Measurements are performed on the volar aspect of the forearm using a Nicolet 800 FTIR spectrometer equipped with an ATR unit with a ZnSe crystal. Hydration of the skin is obtained by occlusion keeping the forearm pressed onto the crystal. Spectra are recorded before and after occlusion up to 30 min. The spectra are fitted with a nonlinear least-squares algorithm with Gaussian bands. Separate band fits of water, normal stratum corneum, and occluded hydrated stratum corneum spectra are obtained yielding band parameters of the individual water contributions in the bending mode at 1640 cm-1, the combination band at 2125 cm-1, and the OH stretches in the hydrated skin stratum corneum spectra. A scaling factor representing the contribution of the water spectrum into the skin stratum corneum spectrum is determined during the occlusion process. In comparison to the dependence of the infrared absorbance ratio in time used by Potts, our scaling factor shows a more distinct transition from enhanced signal due to increased contact area to extra signal due to water content at maximum contact area. Band fit analysis of hydrated skin stratum corneum ATR-FTIR spectra offers the possibility for quantitative determination of individual water band parameters. © 1998 Society of Photo-Optical Instrumentation Engineers.

Non-invasive determination of port wine stain anatomy and physiology for optimal laser treatment strategies.

The treatment of port wine stains (PWSs) using a flashlamp-pumped pulsed dye laser is often performed using virtually identical irradiation parameters. Although encouraging clinical results have been reported, we propose that lasers will only reach their full potential provided treatment parameters match individual PWS anatomy and physiology. The purpose of this paper is to review the progress made on the technical development and clinical implementation of (i) infrared tomography (IRT), optical reflectance spectroscopy (ORS) and optical low-coherence reflectometry (OLCR) to obtain in vivo diagnostic data on individual PWS anatomy and physiology and (ii) models of light and heat propagation, predicting irreversible vascular injury in human skin, to select optimal laser wavelength, pulse duration, spot size and radiant exposure for complete PWS blanching in the fewest possible treatment sessions. Although non-invasive optical sensing techniques may provide significant diagnostic data, development of a realistic model will require a better understanding of relevant mechanisms for irreversible vascular injury.

Changes in optical properties of human whole blood in vitro due to slow heating.

Optical properties of human whole blood were investigated in vitro at 633 nm using a double integrating sphere set-up. The blood flow was maintained at a constant rate through a flow cell while continuously heating the blood at 0.2-1.1 degrees C/min from approximately 25 to 55 degrees C in a heat exchanger. A small, but rather abrupt decrease in the scattering asymmetry factor (g-factor) of 1.7 +/- 0.6% and a similar increase in the scattering coefficient of 2.9 +/- 0.6% were observed at approximately 45-46 degrees C yielding an increase in the reduced scattering coefficient of 40 +/- 10%. Furthermore, a continuous, manifest increase in the absorption coefficient was seen with increasing temperature, on average 80 +/- 70% from 25 to 50 degrees C. The effect of the heating on the blood cells was also studied under a white-light transmission microscope. A sudden change in the shape of the red blood cells, from discshaped to spherical, was observed at approximately the same temperature at which the distinct changes in g-factor and scattering coefficient were observed, i.e. at 45-46 degrees C. The results indicate that this shape transformation could explain the sudden change in scattering properties.

Modelling the assessment of port wine stain parameters from skin surface temperature following a diagnostic laser pulse.

BACKGROUND AND OBJECTIVE: Laser treatment of port wine stains (PWS) has become an established clinical modality over the past decade. However, in some cases full clearance of the PWS cannot be achieved. To improve the clinical results, it is necessary to match the laser treatment parameters to the PWS anatomy on an individual patient basis. Therefore, knowledge of the PWS structure is of great importance. The objective of this study is to describe a diagnostic method to assess the PWS blood vessels depth and diameter from the skin surface temperature-time course following a diagnostic laser pulse. STUDY DESIGN/MATERIALS AND METHODS: The Monte Carlo (MC) method was used to calculate the deposited laser energy into a port wine stain skin model following irradiation by a diagnostic laser pulse at 577 nm. The heat equation was solved numerically, using the deposited energy profile as the source term, yielding the temperature-time course at the skin surface. Subtraction of "bloodless" skin signal from that of the skin containing blood vessels gives us the net contribution of a heated dermal blood vessel to the skin surface temperature-time behaviour. RESULTS: The net blood vessel signal shows heat-diffusion behaviour and was found to be sensitive to the dermal blood vessel depth and diameter. The time delay for the peak signal temperature to occur depends quadratically on the blood vessel depth. The peak temperature relates linearly to the blood vessel diameter. The degree of epidermal melanin content can also be determined from the immediate temperature rise of the signal. CONCLUSION: The proposed method easily enables assessment of the blood vessel depth and diameter as well as the epidermal melanin content in a skin model. The method can be applied to a real PWS when using the adjacent normal skin as a reference.

Modelling light distributions of homogeneous versus discrete absorbers in light irradiated turbid media.

Laser treatment of port wine stains has often been modelled assuming that blood is distributed homogeneously over the dermal volume, instead of enclosed within discrete vessels. The purpose of this paper is to analyse the consequences of this assumption. Due to strong light absorption by blood, fluence rate near the centre of the vessel is much lower than at the periphery. Red blood cells near the centre of the vessel therefore absorb less light than those at the periphery. Effectively, when distributed homogeneously over the dermis, fewer red blood cells would produce the same absorption as the actual number of red blood cells distributed in discrete vessels. We quantified this effect by defining a correction factor for the effective absorbing blood volume of a single vessel. For a dermis with multiple vessels, we used this factor to define an effective homogeneous blood concentration. This was used in Monte Carlo computations of the fluence rate in a homogeneous skin model, and compared with fluence rate distributions using discrete blood vessels with equal dermal blood concentration. For realistic values of skin parameters the homogeneous model with corrected blood concentration accurately represents fluence rates in the model with discrete blood vessels. In conclusion, the correction procedure simplifies the calculation of fluence rate distributions in turbid media with discrete absorbers. This will allow future Monte Carlo computations of, for example, colour perception and optimization of vascular damage by laser treatment of port wine stain models with realistic vessel anatomy.

The effectiveness of massage treatment on cellulite as monitored by ultrasound imaging.

BACKGROUND/AIMS: The visual appearance of cellulite or the'orange peel'look of skin is a common cosmetic problem for many women. Cellulite, or more correctly lipodystrophy, is a modification of the adipose tissue, whereby the fat lobules are swollen as the result of a disturbed blood and lymph micro-circulation and fibrosclerose of connective tissue. In the wealthy diversity of products against cellulite, objective methods to measure their efficacy are of growing importance. The purpose of this study is to establish the effectiveness of a skin massage treatment by quantifying the changes in the skin via ultrasound imaging, during and following treatment. METHODS: Using 20 MHz C-mode ultrasound scanning, a three-dimensional subsurface is constructed that represents the dermis-hypodermis tissue interface. In normal cellulite-free skin, this interface is smooth. In the case of cellulite, however, the dermis-hypodermis junction appears as an irregular surface. Qualitatively, the effect of cellulite treatment is inferred from changes in the shape of this junction. In order to quantify the effect, we chose to monitor the junction area. For the treatments, we used a specially designed handheld electro-mechanical massage device that was moved along the thigh. Treatments were conducted for 3 months, three times a week, during 15 min on each upper leg of 20 healthy female volunteers with moderate symptoms of cellulite (Curri's classification 1-2). Ultrasound measurements were performed monthly, and continued for 2 months after the treatments were stopped. RESULTS: The results, on average, indicate a significant smoothening of the dermis-hypodermis surface (relative surface area reduction 34+3%, 50±3% and 56±2% after 1, 2 and 3 months of treatment, respectively). The smoothening can be described by a mono-exponential function with a time constant of 1.1 month. After the treatments were discontinued, the relative surface area gradually increased (with a time constant of 2.6 month), which indicates that the effect of massage is not permanent. CONCLUSION: Treatment of cellulite by means of an electro-mechanical skin fold massage apparatus significantly smoothens the structure of the dermis-hypodermis interface. Three-dimensional ultrasound imaging of the dermis-hypodermis junction could serve as an objective method to monitor the effectiveness of cellulite treatment.

Time constants in thermal laser medicine: II. Distributions of time constants and thermal relaxation of tissue.

The thermal response of a semi-infinite medium in air, irradiated by laser light in a cylindrical geometry, cannot accurately be approximately by single radial and axial time constants for heat conduction. This report presents an analytical analysis of hear conduction where the thermal response is expressed in terms of distributions over radial and axial time constants. The source term for heat production is written as the product of a Gaussian shaped radial term and an exponentially shaped axial term. The two terms are expanded in integrals over eigenfunctions of the radial and axial parts of the Laplace heat conduction operator. The result is a double integral over the coupled distributions of the two time constants to compute the temperature rise as a function of time and of axial and radial positions. The distribution of axial time constants is a homogeneous slowly decreasing function of spatial frequency (v) indicating that one single axial time constant cannot reasonably characterize axial heat conduction. The distribution of radial time constants is a function centred around a distinguished maximum in the spatial frequency (lambda) close to the single radial time constant value used previously. This suggests that one radial time constant to characterize radial heat conduction may be a useful concept. Special cases have been evaluated analytically, such as short and long irradiation times, axial or radial heat conduction (shallow or deep penetrating laser beams) and, especially, thermal relaxation (cooling) of the tissue. For shallow penetrating laser beams the asymptotic cooling rate is confirmed to be proportional to [(t)0.5-(t-tL)0.5] which approaches 1/t0.5 for t >> tL, where t is the time and tL is the laser pulse duration. For deep penetrating beams this is proportional to 1/(t-tL). For intermediate penetration, i.e. penetration depths about equal to spot size diameters, this is proportional to 1/(t-tL)1.5. The double integral has been evaluated numerically and the results have been compared with the various approximations available including the new results and the single time constant model. The present analysis completes our previous work, presents a closed-form formulation for the non-ablative thermal response of laser irradiated tissue and provides insight into the practical value of using time constants for representing heat conduction effects, in particular for the rate of cooling of the tissue surface.

Light distributions in a port wine stain model containing multiple cylindrical and curved blood vessels.

BACKGROUND AND OBJECTIVES: Knowledge of the light distribution in skin tissue is important for the understanding, prediction, and improvement of the clinical results in laser treatment of port wine stains (PWS). The objective of this study is to improve modelling of PWS treated by laser using an improved and more realistic PWS model. STUDY DESIGN/MATERIALS AND METHODS: Light distributions are calculated by the Monte Carlo method for various PWS blood vessel configurations, such as single and multiple vessels oriented horizontally, curved vessels, and "vertically" oriented vessels. Various vessel sizes and wavelengths are used. RESULTS: Our modelling confirms the concept of selective photothermolysis; 577nm laser light gives maximal deposited energy at the top side of the blood vessels closest to the skin surface and 585nm gives a more uniform energy distribution in the vessel. In the distribution of deposited energy multiple vessels mutually influence each other, because of "shadowing" of diffuse light. CONCLUSION: Modelling PWS laser treatments with multiple vessels confirms the need for successive treatments of vessels layer by layer. The use of different wavelengths affects the local deposited energy profiles in the blood vessels. It predicts that the significance of 585nm laser light lies in the uniform energy distribution in the vessels rather than in gain in energy deposition with depth. The calculated light distributions provide a more realistic input for modelling thermal damage effects in PWS laser treatment and modelling of the epidermal response in thermal imaging of the PWS blood vessel structure.

Laser options for vascular lesions in childhood.

To determine which laser can be used for a vascular lesion, the different working mechanisms of the individual lasers must first be understood. The localization and diameter of the vessels to be treated define whether a certain laser is appropriate for that lesion. For teleangiectasias and port-wine stains the pulsed-dye laser works without scarring. Hemangiomas respond only when flat and superficial. Deeper lesions can only be reached with the ND-Yag laser.

Polarization sensitive coherent anti-Stokes Raman scattering spectroscopy of the amide I band of proteins in solutions.

Polarization sensitive coherent anti-Stokes Raman scattering (PCARS) spectroscopy is a fruitful technique to study Raman vibrations of diluted molecules under off-electron resonant conditions. We apply PCARS as a direct spectroscopic method to investigate the broad amide I band of proteins in heavy water. In spontaneous Raman spectroscopy, this band is not well resolved. We fit a number of spectra taken of each protein under different polarization conditions, with a single set of parameters. It then appears that some substructure is observed in the amide I band. From this substructure, we determine the percentage of alpha-helix, beta-sheet, and random coil for the proteins lysozyme, albumin, ribonuclease A, and alpha-chymotrypsin.

Nonresonant background suppression in CARS spectra of dispersive media using phase mismatching.

A nonresonant background suppression technique using coherent cancellation through phase mismatching is discussed and applied for a noncollinear beam configuration. A cuvette structure consisting of a glass, a sample, and a glass layer is regarded. Phase mismatching is shown to be a useful method to suppress nonresonant contributions from cuvette glass walls as well as those originating from the sample. A numerical calculation reveals a limit for the background suppression which can be achieved with this technique. Measurements using ethanol as a sample show the possibility to compensate the nonresonant background originating from the cuvette walls and to effectively suppress the nonresonant contribution in the spectrum of the sample by a factor of 10-50, yielding Lorentzian bands biased by a constant background. Direct measurement of depolarization ratios without interfering nonresonant background is demonstrated for ethanol and shows that this technique can readily be combined with the polarization sensitive CARS technique.

Compensating pulse-to-pulse fluctuations and increasing spectral reproducibility of phase-matched CARS measurements.

The compensation of pulse-to-pulse fluctuations and the improvement of spectral reproducibility of scanned coherent anti-Stokes Raman scattering (CARS) spectra of dispersing media are discussed. A simple reference CARS setup is presented that needs a minimum number of optical components as a result of using the same optical path for both signals. Variations in spectral profile were found to be caused by mechanical play in the translation stage, which is used to adjust the matching angle. Using retroreflection, the matching angle adjustment is made insensitive to these mechanical imperfections. The multiple interference of probe and signal beam that may occur in thin cuvette walls and its effect on the detected CARS signals are shown, and possible solutions are discussed.