In vitro determination of normal and neoplastic human brain tissue optical properties using inverse adding-doubling

To complement a project towards the development of real-time optical biopsy for brain tissue discrimination and surgical resection guidance, the optical properties of various brain tissues were measured in vitro and correlated to features within clinical diffuse reflectance tissue spectra measured in vivo. Reflectance and transmission spectra of in vitro brain tissue samples were measured with a single-integrating-sphere spectrometer for wavelengths 400-1300 nm and converted to absorption and reduced scattering spectra using an inverse adding-doubling technique. Optical property spectra were classified as deriving from white matter, grey matter or glioma tissue according to histopathologic diagnosis, and mean absorption and reduced scattering spectra were calculated for the three tissue categories. Absolute reduced scattering and absorption values and their relative differences between histopathological groups agreed with previously reported results with the exception that absorption coefficients were often overestimated, most likely due to biologic variability or unaccounted light loss during reflectance/transmission measurement. Absorption spectra for the three tissue classes were dominated by haemoglobin absorption below 600 nm and water absorption above 900 nm and generally determined the shape of corresponding clinical diffuse reflectance spectra. Reduced scattering spectral shapes followed the power curve predicted by the Rayleigh limit of Mie scattering theory. While tissue absorption governed the shape of clinical diffuse reflectance spectra, reduced scattering determined their relative emission intensities between the three tissue categories.     

In vivo optical characterization of human tissues from 610 to 1010 nm by time-resolved reflectance spectroscopy

A fully automated system for time-resolved reflectance spectroscopy based on tunable mode-locked laser sources and on time-correlated single-photon counting for the detection of time-resolved reflectance data was applied to the evaluation of the optical properties of biological tissues (arm, abdomen and forehead) in vivo from 610 to 1010 nm. The scattering decreases progressively with increasing wavelength, while the absorption line shapes show the typical spectral features of the principal tissue components (haemoglobin, water and lipid), with different weights depending on the tissue type. The best fit of the absorption spectra measured in vivo with the spectra of the pure constituents yielded information on the percentage composition of the different tissues. The interpretation of transport scattering spectra with Mie theory provided information on tissue structure.     

A simple algorithm for in vivo ocular fundus oximetry compensating for non-haemoglobin absorption and scattering

An algorithm is introduced for the compensation of the influence of non-haemoglobin absorption as well as tissue scattering on blood spectra used in optical oximetry at the ocular fundus. The in vivo measured spectra were corrected by a linear transformation in order to match the reference spectra of fully oxygenated and reduced blood, respectively, at three isosbestic points (522 nm, 569 nm and 586 nm). The oxygen saturation can then be determined at a wavelength showing a high contrast between oxygenated and reduced haemoglobin (e.g., 560 nm). Reflection measurements at blood flowing through cuvettes were used to validate the algorithm. The oxygen saturation values were compared to measurements of the same samples at a laboratory haemoximeter. The mean deviation was found to be 2.65%.     

Optical biopsy of bone tissue: a step toward the diagnosis of bone pathologies

In vivo absorption and reduced scattering spectra of the human calcaneous from 650 to 1000 nm were assessed using a laboratory system for time-resolved transmittance spectroscopy. Measurements were performed on the calcaneous of seven female volunteers ranging from 26 to 82 years of age. The analysis of the absorption spectra, using a linear combination of the key tissue absorbers (bone mineral, water, lipids, oxy- and deoxyhemoglobin), revealed a general decrease in bone mineral content and an increase in lipids with age, which is in agreement with the aging transformations that occur in bone tissues. The scattering spectra were less effective in detecting such changes in older subjects, showing only a minor decrease in the coefficient for these subjects. The capability to noninvasively quantify bone tissue composition suggests a possible use of optical biopsy for the diagnosis of bone pathologies such as osteoporosis, which are characterized by a progressive reduction and transformation of the mineral in the bone matrix.     

Thermal coagulation-induced changes of the optical properties of normal and adenomatous human colon tissues in vitro in the spectral range 400-1,100 nm

The absorption coefficients, the reduced scattering coefficients and the optical penetration depths for native and coagulated human normal and adenomatous colon tissues in vitro were determined over the range of 400-1,100 nm using a spectrophotometer with an internal integrating sphere system, and the inverse adding-doubling method was applied to calculate the tissue optical properties from diffuse reflectance and total transmittance measurements. The experimental results showed that in the range of 400-1,100 nm there were larger absorption coefficients (P < 0.01) and smaller reduced scattering coefficients (P < 0.01) for adenomatous colon tissues than for normal colon tissues, and there were smaller optical penetration depths for adenomatous colon tissues than for normal colon tissues, especially in the near-infrared wavelength. Thermal coagulation induced significant increase of the absorption coefficients and reduced scattering coefficients for the normal and adenomatous colon tissues, and significantly reduced decrease of the optical penetration depths for the normal and adenomatous colon tissues. The smaller optical penetration depth for coagulated adenomatous colon tissues is a disadvantage for laser-induced thermotherapy (LITT) and photodynamic therapy (PDT). It is necessary to adjust the application parameters of lasers to achieve optimal therapy.     

Time-domain scanning optical mammography: II. Optical properties and tissue parameters of 87 carcinomas

Within a clinical trial on scanning time-domain optical mammography reported on in a companion publication (part I), craniocaudal and mediolateral projection optical mammograms were recorded from 154 patients, suspected of having breast cancer. Here we report on in vivo optical properties of the subset of 87 histologically validated carcinomas which were visible in optical mammograms recorded at two or three near-infrared wavelengths. Tumour absorption and reduced scattering coefficients were derived from distributions of times of flight of photons recorded at the tumour site employing the model of diffraction of photon density waves by a spherical inhomogeneity, located in an otherwise homogeneous tissue slab. Effective tumour radii, taken from pathology, and tumour location along the compression direction, deduced from off-axis optical scans of the tumour region, were included in the analysis as prior knowledge, if available. On average, tumour absorption coefficients exceeded those of surrounding healthy breast tissue by a factor of about 2.5 (670 nm), whereas tumour reduced scattering coefficients were larger by about 20% (670 nm). From absorption coefficients at 670 nm and 785 nm total haemoglobin concentration and blood oxygen saturation were deduced for tumours and surrounding healthy breast tissue. Apart from a few outliers total haemoglobin concentration was observed to be systematically larger in tumours compared to healthy breast tissue. In contrast, blood oxygen saturation was found to be a poor discriminator for tumours and healthy breast tissue; both median values of blood oxygen saturation are the same within their statistical uncertainties. However, the ratio of total haemoglobin concentration over blood oxygen saturation further improves discrimination between tumours and healthy breast tissue. For 29 tumours detected in optical mammograms recorded at three wavelengths (670 nm, 785 nm, 843 nm or 884 nm), scatter power was derived from transport scattering coefficients. Scatter power of tumours tends to be larger than that of surrounding healthy breast tissue, yet the 95% confidence intervals of both medians overlap.     

蛋清热变过程中的光学特性变化

目的为解决手术中激光切割能力下降的问题,以鸡蛋蛋清为样本,研究热变过程中生物组织的光学特性变化。方法使用积分球系统测量650nm激光照射下的样本在热风枪加热下,从26％升温到99℃过程中的准直透射光、漫透射光和反射光光强随温度变化的数据,计算吸收系数和散射系数变化曲线。结果样本热变前光学特性受温度影响较小,热变过程中透射率降低,吸收系数和散射系数增大。结论手术中低强度激光照射组织发生热变使吸收能力下降,应保持较高激光强度以达到消融阈值。     

Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions

The absorption coefficient mu(a), scattering coefficient mu(s), and anisotropy factor g of diluted and undiluted human blood (hematocrit 0.84 and 42.1%) are determined under flow conditions in the wavelength range 250 to 1100 nm, covering the absorption bands of hemoglobin. These values are obtained by high precision integrating sphere measurements in combination with an optimized inverse Monte Carlo simulation (IMCS). With a new algorithm, appropriate effective phase functions could be evaluated for both blood concentrations using the IMCS. The best results are obtained using the Reynolds-McCormick phase function with the variation factor alpha = 1.2 for hematocrit 0.84%, and alpha = 1.7 for hematocrit 42.1%. The obtained data are compared with the parameters given by the Mie theory. The use of IMCS in combination with selected appropriate effective phase functions make it possible to take into account the nonspherical shape of erythrocytes, the phenomenon of coupled absorption and scattering, and multiple scattering and interference phenomena. It is therefore possible for the first time to obtain reasonable results for the optical behavior of human blood, even at high hematocrit and in high hemoglobin absorption areas. Moreover, the limitations of the Mie theory describing the optical properties of blood can be shown.     

Optical properties of normal and diseased human breast tissues in the visible and near infrared

The optical absorption and scattering coefficients have been determined for specimens of normal and diseased human breast tissues over the range of wavelengths from 500 to 1100 nm. Total attenuation coefficients were measured for thin slices of tissue cut on a microtome. The diffuse reflectance and transmittance were measured for 1.0 mm thick samples of these tissues, using standard integrating sphere techniques. Monte Carlo simulations were performed to derive the scattering and absorption coefficients, as well as the mean cosine of the scattering angle. The results indicate that scatter exceeds absorption by at least two orders of magnitude. Absorption is most significant at wavelengths below 600 nm. The scattering coefficients lie in the range 30-90 mm-1 at 500 nm, and fall smoothly with increasing wavelength to between 10 and 50 mm-1 at 1100 nm. The scattering coefficient for adipose tissue differs, in that it is invariant with wavelength over this spectral range. For all tissues examined, the scattered light is highly forward peaked, with the mean cosine of the scattering angle in the range 0.945-0.985. Systematic differences between the optical properties of some tissue types are demonstrated.     

Optical properties of porcine skin dermis between 900 nm and 1500 nm

The weak absorption of shortwave infrared light by skin tissues between 700 and 1500 nm offers an important window for diagnosis by optical means. The strong scattering of shortwave infrared light by the skin, however, presents a challenge to the modelling of light propagation through the skin and the understanding of skin optics. We have measured the collimated and diffuse transmittance and diffuse reflectance of porcine skin dermis samples within 30 h post-mortem. Monte Carlo simulations have been performed to inversely determine the absorption coefficient, scattering coefficient and anisotropy factor of the dermis samples in the spectral range from 900 to 1500 nm. We further analyse the sensitivity of the values of the parameters to the experimental errors and inverse calculation procedures. The state of the cellular integrity of the skin samples following optical measurements was verified using transmission electron microscopy. These results were correlated to study post-mortem effects on the in vitro optical properties of porcine dermis. We concluded that for samples stored within crushed ice for up to 30 h post-mortem the wavelength dependence of optical properties of the dermis remains unchanged while the values of the parameters vary moderately due to modification of the water content of the tissue.     

In-vivo characterization of optical properties of pigmented skin lesions including melanoma using oblique incidence diffuse reflectance spectrometry

In this letter, we report the first use of oblique incidence diffuse reflectance spectrometry to conduct in-vivo measurements of optical properties of three different types of pigmented skin lesions, including melanoma, dysplastic, and common nevi. Both absorption and reduced scattering coefficient spectra were estimated from the spatially resolved diffuse reflectance within the wavelength range of 455-765 nm for 144 pigmented skin lesions including 16 melanomas. The absorption and reduced scattering spectra were found to change with the malignancy of the skin lesions, which were generally higher for the malignant cases than the benign ones. Based on the measurement results, the physiological origin leading to the change of the absorption and scattering properties is also discussed.     

Optical characterization of a radiochromic film by total reflectance and transmittance measurements

The GafChromic film (GCF) MD-55-2, a radiochromic material, was examined for its optical properties through total reflectance and transmittance measurements in visible spectrum (400-700 nm). By using a multilayer model of the film and Kubelka-Munk's (KM) theory, absorption and scattering coefficients of the film sensitive layer (K and S, respectively) were obtained from measurements of irradiated and nonirradiated slides. This has allowed calculation of the absorbance A(KM) of the sensitive layer of the GCF. The model easily splits scattering from absorption. Unlike absorption, scattering is essentially insensitive to irradiation dose and decreases slowly as the wavelength increases. The scattering effect is predominant over absorption in the 400-500 nm range, while beyond 600 nm absorption prevails. The A(KM) absorbance of the sensitive layer was calculated using the K coefficient and compared with the optical densities (OD) measured considering only ballistic photons (as in a standard spectrophotometer) as well as the optical densities measured collecting all the transmitted photons (as in many densitometers). The values of A(KM) found were always lower than OD measured by the other methods and they had the best linearity on the whole visible range. These data support the hypothesis that the sensitive layer reacts to irradiation more linearly than that shown by measurements using standard commercial devices. However, in the 600-680 nm range, correction is not very important because absorption is predominant over scattering. When GCF is used for imaging, scattering produces a loss of spatial information. Consequently, it is necessary to collect only ballistic photons and to correct absorbance by K and S coefficients.     

Reconstruction of optical properties of phantom and breast lesion in vivo from paraxial scanning data

We report on the reconstruction of absorption and reduced scattering coefficients of breast tissue in vivo of a patient with mastopathic disease. Distributions of times of flight of photons through the compressed breast were recorded by paraxial scanning. From data measured at four different source-detector offsets optical properties were reconstructed within the linear Rytov approximation by a fast inverse Fourier space method. Low-pass filtering in Fourier space was employed to remove excessive noise from high spatial frequency components and to reduce the computational efforts by a factor of 3, typically. The mammograms displaying reconstructed absorption and reduced scattering coefficients were compared with projection mammograms either obtained by time-window analysis of experimental data or based on average absorption and reduced scattering coefficients which were derived from measured temporal point spread functions within a simple homogeneous model. All inhomogeneities which were visible in the projection mammograms and which could be associated with specific breast tissue compartments could be correlated with inhomogeneities in the reconstructed absorption coefficient. In particular, the mastopathic disease was detected in the reconstructed absorption mammogram. In order to assess reliability of optical properties reconstructed from data obtained by paraxial scanning, corresponding phantom experiments and reconstructions of phantom optical properties were carried out. Because of the limited angular range sampled by the in vivo and phantom measurements, considerable blurring of the absorption coefficient occurs along the compression direction, compromising longitudinal resolution.     

In vivo determination of optical properties of normal and tumor tissue with white light reflectance and an empirical light transport model during endoscopy

Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87+/-0.22 cm(-1) and 7.8+/-2.3 cm(-1), respectively. Cancerous mucosa had values 1.87+/-1.10 cm(-1) and 8.4+/-2.3 cm(-1), respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues.     

Estimation of subcellular particle size histograms with electron microscopy for prediction of optical scattering in breast tissue

Diffuse near-infrared tomography of tissue reveals scattering changes that originate from the submicroscopic features of the tissue; yet the existing tools to use this information to predict which features contribute to the scattering spectrum are limited by the lack of direct data quantifying the particle sizes. Breast tissue was examined with electron microscopy, and analysis showed that the distributions of particle sizes appear in double exponential functions for most cellular tissues. The average particle size histograms of high-grade cancer, low-grade cancer, fibroglandular tissue, and adipose tissue were examined. The particle histograms were progressively decreasing in magnitude for these tissue types, and the average size of the particles increased, for these four tissues, respectively. Typical particle sizes in the range of 10 to 500 nm for these tissue types, with biexponential fitting, gave two particle distributions: one near 20 to 25 nm for the smaller size and one at 110 to 230 nm for the larger distributions. Mie scatter theory was used to take these particle distributions and calculate scattering spectra. The ability to image reduced scattering coefficient spectra of bulk breast tissues exists, and so this data provides insight into how bulk imaging may be mapped over to predict factors related to the tissue ultrastructure.     

In vivo endoscopic tissue diagnostics based on spectroscopic absorption,scattering, and phase function properties

A fast spectroscopic system for superficial and local determination of the absorption and scattering properties of tissue (480 to 950 nm) is described. The probe can be used in the working channel of an endoscope. The scattering properties include the reduced scattering coefficient and a parameter of the phase function called gamma, which depends on its first two moments. The inverse problem algorithm is based on the fit of absolute reflectance measurements to cubic B-spline functions derived from the interpolation of a set of Monte Carlo simulations. The algorithm's robustness was tested with simulations altered with various amounts of noise. The method was also assessed on tissue phantoms of known optical properties. Finally, clinical measurements performed endoscopically in vivo in the stomach of human subjects are presented. The absorption and scattering properties were found to be significantly different in the antrum and in the fundus and are correlated with histopathologic observations. The method and the instrument show promise for noninvasive tissue diagnostics of various epithelia.     

Experimental and numerical study of the colour appearance of tattoo models

The colour of tattooed skin has been predicted by a Monte Carlo method based on the optical coefficient spectra of the skin and tattoo dyes. Slices of pig skin, a tattoo phantom and skin phantoms with different thickness were prepared, and their reflectance and transmittance spectra were measured using an integrating sphere at wavelengths varying from 400 nm to 700 nm. The absorption and scattering coefficient spectra of skin phantoms, pig skins and the tattoo phantom were each calculated using the inverse Monte Carlo method. The skin phantoms and pig skins were overlaid on the tattoo phantom, and the reflectance spectra of the two-layered structures were measured. The reflectance spectra of the two-layered structures were calculated from the optical coefficient spectra using the Monte Carlo method. They agreed well with the measured spectra. The colour differences between the calculated and measured spectra were also evaluated by the L^*a^*b^* colour space distances and showed good agreement, with 3.49 for the skin phantoms and 8.27 for the pig skins.     

Influence of shear rate on the optical properties of human blood in the spectral range 250 to 1100 nm

The intrinsic optical parameters-absorption coefficient mua, scattering coefficient mus, anisotropy factor g, and effective scattering coefficient mus'--are determined for human red blood cells of hematocrit 42.1% dependent on the shear rate in the wavelength range 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulation are carried out for different wall shear rates between 0 and 1000 s(-1). Randomly oriented cells show maximal mua, mus, and mus' values. Cell alignment and elongation, as well as the Fahraeus effect at increasing shear rates, lead to an asymptotical decrease of these values. The anisotropy factor shows this behavior only below 600 nm, dependent on absorption; above 600 nm, g is almost independent of shear rate. The decrease of mus' is inversely correlated with the hemoglobin absorption. Compared to randomly oriented cells, aggregation reduces all parameters by a different degree, depending on the hemoglobin absorption. It is possible to evaluate the influence of collective scattering phenomena, the absorption within the cell, and the cell shape.     

Determination of the optical properties of the human uterus using frequency-domain photon migration and steady-state techniques

The optical properties (absorption and transport scattering coefficients) of freshly excised, bulk human uterine tissues were measured at 630 nm using frequency-domain and steady-state photon migration techniques. Measurements were made on both normal (pre- and post-menopausal) and non-neoplastic fibrotic tissues. The absorption coefficient of normal post-menopausal tissue (approximately 0.06 mm(-1)) was found to be significantly greater than that of normal pre-menopausal tissue (0.02-0.03 mm(-1)) and pre-menopausal fibrotic tissue (0.008 mm(-1)). The transport scattering coefficient was similar in all three tissue types considered (0.6-0.9 mm(-1)). From the preliminary results presented here, we conclude that optical properties can be reliably calculated either from the frequency-dependent behaviour of diffusely propagating photon density waves or by combining the frequency-independent photon density wave phase velocity with steady-state light penetration depth measurements. Instrument bandwidth and tissue absorption relaxation time ultimately determine the useful frequency range necessary for frequency-domain photon migration (FDPM) measurements. Based on the optical properties measured in this study, we estimate that non-invasive FDPM measurements of normal uterine tissue require modulation frequencies in excess of 350 MHz.