Colorectal cancer is the second most common cancer and the second with the highest associated deaths in the world. Methods used in clinical practice for colon cancer diagnosis are fairly effective but quite unpleasant and not always applicable in situations where the patient has symptoms of colonic obstruction. This problem can be solved by the use of optical methods that can be applied less invasively.
This study presents the results of classification of cancerous and healthy colon tissue absorption coefficient spectra. The absorption coefficient was measured using direct calculations from the total reflectance and total transmittance spectra obtained ex vivo. Classification was performed using support vector machine, multilayer perceptron and linear discriminant analysis.
The concept of ‘tissue optical windows’ and method of optical clearing (OC) based on controllable and reversible modification of tissue optical properties by their soaking with a biocompatible optical clearing agent (OCA) are prsented. Fundamentals and major mechanisms of OC allowing one to enhance optical imaging facilities and laser treatment efficiency of living tissues are described. Perspectives of immersion optical clearing/contrasting technique aiming to enhance optical imaging of living tissues by using different imaging modalities working in the ultra-broad wavelength range from deep UV to terahertz waves are discussed. It demonstrated that immersion OC method can be applied to evaluate the characteristic diffusion properties of water and OCA in various tissues and even discriminate between the mobile water content in normal and pathological tissues.
The interest of using light in clinical practice is increasing strongly and many applications work at various wavelengths from the ultraviolet to the infrared. Due to this great range of applications, the determination of the optical properties of biological tissues in a wide spectral range becomes of interest. The liver is an important organ, since it has a major role in the human body and various pathologies are known to develop within it. For these reasons, this study concerns the estimation of the optical properties of human normal and pathological (metastatic carcinoma) liver tissues between 200 and 1000 nm. The obtained optical properties present the expected wavelength dependencies for both tissues – the refractive index, the absorption and the scattering coefficients decrease with the wavelength and the anisotropy and light penetration depth increase with the wavelength. Although similar behavior was observed for the various properties between the normal and pathological tissues, evidence of smaller blood content in the pathological tissues was found. A possible explanation is that the cancer cells destroy liver’s vasculature and internal architecture, providing though a reduction in the blood content. For low wavelengths, it was observed a matching between the scattering and the reduced scattering coefficients, which implies a nearly zero anisotropy in that range. The scattering coefficient decreases from nearly 140 cm-1 (at 200 nm) to 80 cm-1 (at 1000 nm) for the normal liver and from nearly 140 cm-1 (at 200 nm) to 95 cm-1 (at 1000 nm) for the pathological tissue.
To characterize the optical clearing treatments in human colorectal tissues and possibly to differentiate between treatments of normal and pathological tissues, we have used a simple indirect method derived from Mie scattering theory to estimate the kinetics of the reduced scattering coefficient. A complementary method to estimate the kinetics of the scattering coefficient is also used so that the kinetics of the anisotropy factor and of the refractive index are also calculated. Both methods rely only on the thickness and collimated transmittance measurements made during treatment. The results indicate the expected time dependencies for the optical properties of both tissues: an increase in the refractive index and anisotropy factor and a decrease in the scattering coefficients. The similarity in the kinetics obtained for normal and pathological tissues indicates that optical clearing treatments can be applied also in pathological tissues to produce similar effects. The estimated time dependencies using experimental spectral data in the range from 400 to 1000 nm allowed us to compare the kinetics of the optical properties between different wavelengths.
Knowledge of the optical properties of tissues is necessary, since they change from tissue to tissue and can differ between normal and pathological conditions. These properties are used in light transport models with various areas of application. In general, tissues have significantly high scattering coefficient when compared to the absorption coefficient and such difference usually increases with decreasing wavelength. The study of the wavelength dependence of the optical properties has been already made for several animal and human tissues, but extensive research is still needed in this field. Considering that most of the Biophotonics techniques used in research and clinical practice use visible to NIR light, we have estimated the optical properties of colorectal muscle (muscularis propria) between 400 and 1000 nm. The samples used were collected from patients undergoing resection surgery for colorectal carcinoma. The estimated scattering coefficient for colorectal muscle decreases exponentially with wavelength from 122 cm-1 at 400 nm to 95 cm-1 at 650 nm and to 91 cm-1 at 1000 nm. The absorption coefficient shows a wavelength dependence according to the behavior seen for other tissues, since it decreases from 8 cm-1 at 400 nm to 2.6 cm-1 at 650 nm and to 1.3 cm-1 at 1000 nm. The estimated optical properties differ from the ones that we have previously obtained for normal and pathological colorectal mucosa. The data obtained in this study covers an extended spectral range and it can be used for planning optical clearing treatments for some wavelengths of interest.
The optical dispersion and water content of human liver were experimentally studied to estimate the optical dispersions of tissue scatterers and dry matter. Using temporal measurements of collimated transmittance [Tc(t)] of liver samples under treatment at different glycerol concentrations, free water and diffusion coefficient (Dgl) of glycerol in liver were found as 60.0% and 8.2×10−7 cm2/s, respectively. Bound water was calculated as the difference between the reported total water of 74.5% and found free water. The optical dispersion of liver was calculated from the measurements of refractive index (RI) of tissue samples made for different wavelengths between 400 and 1000 nm. Using liver and water optical dispersions at 20°C and the free and total water, the dispersions for liver scatterers and dry matter were calculated. The estimated dispersions present a decreasing behavior with wavelength. The dry matter dispersion shows higher RI values than liver scatterers, as expected. Considering 600 nm, dry matter has an RI of 1.508, whereas scatterers have an RI of 1.444. These dispersions are useful to characterize the RI matching mechanism in optical clearing treatments, provided that [Tc(t)] and thickness measurements are performed during treatment. The knowledge of Dgl is also important for living tissue cryoprotection applications.
Optical properties of biological tissues are unique and may be used for tissue identification, tissue discrimination or even to identify pathologies. Early stage colorectal cancer evolves from adenomatous polyps that arise in the inner layer of the colorectal tube — the mucosa. The identification of different optical properties between healthy and pathological colorectal tissues might be used to identify different tissue components and to develop an early stage diagnosis method using optical technologies. Since most of the biomedical optics techniques use light within the visible and near infrared wavelength ranges, we used the inverse adding-doubling method to make a fast estimation of the optical properties of colorectal mucosa and early stage adenocarcinoma between 400 and 1000 nm. The estimated wavelength dependencies have provided information about higher lipid content in healthy mucosa and higher blood content in pathological tissue. Such data has also indicated that the wavelength dependence of the scattering coefficient for healthy mucosa is dominated by Rayleigh scattering and for pathological mucosa it is dominated by Mie scattering. Such difference indicates smaller scatterer size in healthy mucosa tissue. Such information can now be used to develop new diagnosis or treatment methods for early cancer detection or removal. One possibility is to use optical clearing technique to improve tissue transparency and create localized and temporary tissue dehydration for image contrast improvement during diagnosis or polyp laser removal. Such techniques can now be developed based on the different results that we have found for healthy and pathological colorectal mucosa.
Colorectal carcinoma is a major health concern worldwide and its high incidence and mortality require accurate screening methods. Following endoscopic examination, polyps must be removed for histopathological characterization. Aiming to contribute to the improvement of current endoscopy methods of colorectal carcinoma screening or even for future development of laser treatment procedures, we studied the diffusion properties of glucose and water in colorectal healthy and pathological mucosa. These parameters characterize the tissue dehydration and the refractive index matching mechanisms of optical clearing (OC). We used ex vivo tissues to measure the collimated transmittance spectra and thickness during treatments with OC solutions containing glucose in different concentrations. These time dependencies allowed for estimating the diffusion time and diffusion coefficient values of glucose and water in both types of tissues. The measured diffusion times for glucose in healthy and pathological mucosa samples were 299.2±4.7 s and 320.6±10.6 s for 40% and 35% glucose concentrations, respectively. Such a difference indicates a slower glucose diffusion in cancer tissues, which originate from their ability to trap far more glucose than healthy tissues. We have also found a higher free water content in cancerous tissue that is estimated as 64.4% instead of 59.4% for healthy mucosa.
Part of the optical clearing study in biological tissues concerns the determination of the diffusion characteristics of water and optical clearing agents in the subject tissue. Such information is sufficient to characterize the time dependence of the optical clearing mechanisms—tissue dehydration and refractive index (RI) matching. We have used a simple method based on collimated optical transmittance measurements made from muscle samples under treatment with aqueous solutions containing different concentrations of ethylene glycol (EG), to determine the diffusion time values of water and EG in skeletal muscle. By representing the estimated mean diffusion time values from each treatment as a function of agent concentration in solution, we could identify the real diffusion times for water and agent. These values allowed for the calculation of the correspondent diffusion coefficients for those fluids. With these results, we have demonstrated that the dehydration mechanism is the one that dominates optical clearing in the first minute of treatment, while the RI matching takes over the optical clearing operations after that and remains for a longer time of treatment up to about 10 min, as we could see for EG and thin tissue samples of 0.5 mm.
To determine the differences between the optical clearing effects created by ethylene glycol in fresh and frozen samples, we have performed several measurements from samples in both conditions. Fresh samples were used after animal sacrifice and frozen samples were kept at -20°C for 72 hours. The different measurements performed with samples from both cases were total transmittance, collimated transmittance, total reflectance and specular reflectance. Considering, for instance, collimated transmittance measurements, we have verified that the spectra measured from both samples before adding the solution present different levels of collimated transmittance. The time-dependence evolution of the collimated transmittance spectrum is similar between both cases of samples, but since they present different levels of “natural” transmittance, the optical clearing effect is observed at different levels if we compare between fresh and frozen samples.
Optical characterization and internal structure of biological tissues is highly important for biomedical optics. In particular
for optical clearing processes, such information is of vital importance to understand the mechanisms involved through
the variation of the refractive indices of tissue components. The skeletal muscle presents a fibrous structure with an
internal arrangement of muscle fiber cords surrounded by interstitial fluid that is responsible for strong light scattering.
To determine the refractive index of muscle components we have used a simple method of measuring tissue mass and
refractive index during dehydration. After performing measurements for natural and ten dehydration states of the muscle
samples, we have determined the dependence between the refractive index of the muscle and its water content. Also, we
have joined our measurements with some values reported in literature to perform some calculations that have permitted
to determine the refractive index of the dried muscle fibers and their corresponding volume percentage inside the natural
muscle.
It is known that the fibrous structure of muscle causes light scattering. This phenomenon occurs due to the refractive index discontinuities located between muscle fibers and interstitial fluid. To study the possibility of reducing light scattering inside muscle, we consider its spectral transmittance evolution during an immersion treatment with an optical clearing solution containing ethanol, glycerol, and distilled water. Our methodology consists of registering spectral transmittance of muscle samples while immersed in that solution. With the spectral data collected, we represent the transmittance evolution for some wavelengths during the treatment applied. Additionally, we study the variations that the treatment has caused on the samples regarding tissue refractive index and mass. By analyzing microscopic photographs of tissue cross section, we can also verify changes in the internal arrangement of muscle fibers caused by the immersion treatment. Due to a mathematical model that we develop, we can explain the variations observed in the studied parameters and estimate the amount of optical clearing agent that has diffused into the tissue samples during the immersion treatment. At the end of the study, we observe and explain the improvement in tissue spectral transmittance, which is approximately 65% after 20 min.
Skeletal muscle presents an internal fibrous structure. The existence of muscle fibers surrounded by interstitial fluid
originates an internal step refractive index profile that causes light scattering. One way to minimize this effect inside a
muscle is to perform an optical clearing treatment, using an adequate solution that presents a refractive index higher than
the interstitial fluid. We have studied muscle spectral transmittance during sample immersion in propylene glycol. With
the collection of transmittance spectra registered during a period of 20 minutes of immersion we could represent spectral
transmittance evolution for several wavelengths and verify that the tissue samples have become more translucent. The
optical clearing effect created in the tissue samples was characterized by an increase of 45% above the natural
transmittance and the variations observed in tissue mass, pH and global refractive index. We also identified the initial
mechanisms of agent diffusion into the tissue and consequent tissue dehydration from the spectral transmittance
evolution. The histological analysis of variations caused in the internal structure of the tissues permitted to better explain
the optical clearing effect created. Considering a mathematical model developed in previous studies, we could estimate
the amount of agent that was inserted into the tissue samples.
Skeletal muscle is a fibrous tissue composed by muscle fibers and interstitial fluid. Due to this constitution, the muscle
presents a non uniform refractive index profile that origins strong light scattering. One way to improve tissue
transmittance is to reduce this refractive index mismatch by immersing the muscle in an optical clearing agent. As a
consequence of such immersion tissue also suffers dehydration. The study of the optical clearing effect created by a
simple mixture composed by ethanol, glycerol and distilled water has proven its effectiveness according to the variations
observed in the parameters under study. The effect was characterized in terms of its magnitude, time duration and
histological variations. The applied treatment has created a small reduction of the global sample refractive index that is
justified by the long time rehydration caused by water in the immersing solution. From the reduction in sample pH we
could also identify the dehydration process created in the sample. The immersion treatment has originated fiber bundle
contraction and a spread distribution of the muscle fiber bundles inside. New studies with the mixture used, or with other
combinations of its constituents might be interesting to perform with the objective to develop new clinical procedures.
We intended to characterize and compare the dependence between the concentration of two optical clearing agents and
the effects created by them in muscle from rat. Using Ethylene glycol and Glycerol in three distinct concentrations, we
expected to measure time evolution of the optical transmittance and variations created in tissue samples regarding mass,
pH, thickness and histological parameters. Measuring natural state properties of tissue, we establish reference parameters
to quantify variations in samples due to osmotic immersion treatment. Such variations were correlated with the optical
clearing effect created in tissue and identified with time evolution of sample transmission. We observed for all the
samples and agents studied that tissue transmission rises in time during the treatment with the osmotic solutions. Also,
tissue thickness and refractive index show an increase, while the sample's pH lowers due to water loss inside tissue
samples. Muscle fibres become more spatially separated after treatment due to osmotic impregnation inside the
interstitial space. The variations described are stronger as the solution's concentration becomes higher. By comparing
between results obtained with solutions of Ethylene glycol and Glycerol in the same concentration, we could verify
similar effects but stronger when the Glycerol solution was applied.
Computational methods have been used with great application to biomedical optics. The events created by the interaction of radiation with biological materials can easily be translated to computer languages with the objective of producing simulation techniques to be used prior to physical intervention. The addition of biocompatible and hyper osmotic agents to several types of biological tissues has proven the enhancement of transparency to radiation flux by reduction of material's optical properties. The evolutionary behavior of the agent's action in the tissue samples before saturation has been observed by numerous researchers but has never been described mathematically. In the present work we will describe the application of Monte Carlo simulation to estimate the evolutionary states of optical transparency of biological tissues when immersed in an osmotic solution. We begin our study with typical values for the optical properties of rabbit muscle and proceed by reducing the absorption and scattering coefficients independently and simultaneously. The results show the number of transmitted, absorbed, scattered and reflected photons in different stages of the action of a generic osmotic agent over a small and well defined tissue sample.
Port Wine ageing process is very important to produce the most appreciated and expensive wines from the class. The process takes decades to accomplish and involves particular techniques which are taken inside refrigerated cellars. Different wines pass through such process to produce 10 year, 20 year, 30 year and 40 year Ports. There are no documented data about color or turbidity evolution during the ageing process. We decided to verify the states of color and spectral turbidity of different aged Gold white port wine. The acquired results show a spectral evolution on transmition and scattered radiation along with color modification which are a close and direct consequence of adopted corrective measures. In measuring the four samples, we have used our spectronephelometer with optical fiber tips to illuminate sample and to acquire transmitted or scattered radiation. Transmition results were calibrated with a standard spectrophotometer at our laboratory, and scattered spectra were measured considering a system calibration with ISO12103 standard dust. We are aware that the four samples were harvested in different years, but the wine type is the same and the ageing process does not differ from one sample to another.
Spectronephelometric measurement techniques are in the order of the day. We can apply these techniques to monitor the production of consumable fluids and to verify their quality. Products like Wine, Beer and Olive Oil for instance, are widely consumed over the world. These products do have a major role in people’s dietary habits and their quality is of greater concern from day to day. If we can make use of a monitoring system that is able to perform measurements in situ, on line and in real time, then we will obviously have the capacity to improve quality. Particles that are suspended in consumable fluid samples interact with radiation by scattering it in almost all directions. If we can detect this scattered radiation, then we have information on the suspended particles. Making use on some Physical relations, we can transpose this information to physical parameters like Color and Turbidity.
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