Luís Oliveira

Optical measurements of rat muscle samples under treatment with ethylene glycol and glucose

With the objective to study the variation of optical properties of rat muscle during optical clearing, we have performed a set of optical measurements from that kind of tissue. The measurements performed were total transmittance, collimated transmittance, specular reflectance and total reflectance. This set of measurements is sufficient to determine diffuse reflectance and absorbance of the sample, also necessary to estimate the optical properties. All the performed measurements and calculated quantities will be used later in inverse Monte Carlo (IMC) simulations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose. The results obtained with the measurements already provide some information about the optical clearing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.

Diffusion characteristics of ethylene glycol in skeletal muscle

Abstract. 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.

Rat muscle opacity decrease due to the osmosis of a simple mixture

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.

Port wine spectronephelometry

The main purpose of this research was to create a portable equipment capable of measuring colour and turbidity during production of port wine. Actually, human, chemical and colour analysis are performed in key production stages for quality control. Several wine transformation processes are done without any kind of control. By using a fibre optic illuminator with a tungsten/halogen light source and a small spectrometer with fibre optic input, it was possible to perform angular measurements in wine samples. We have adapted these optical devices with a specially designed mechanical equipment which allows the passage of the wine during production. The spectral measurements were acquired in four different angular positions in a bypass of the mechanical support. This way we could determine colour coordinates and spectral transmittance and turbidity for three stages of a Ruby production. The entire equipment assembly had been previously calibrated in terms of colour and turbidity determination. The obtained results are in agreement with wine transformations between measurements, showing a small variation in colour and a high diminishing in spectral turbidity when filtration was performed. Transmittance has also increased after filtration turning the wine more limpid as it should be for human appreciation.

Glucose diffusion in colorectal mucosa—a comparative study between normal and cancer tissues

Abstract. 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±4.7u2009u2009s and 320.6±10.6u2009u2009s 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.

Optical clearing mechanisms characterization in muscle

Optical immersion clearing is a technique that has been widely studied for more than two decades and that is used to originate a temporary transparency effect in biological tissues. If applied in cooperation with clinical methods it provides optimization of diagnosis and treatment procedures. This technique turns biological tissues more transparent through two main mechanisms — tissue dehydration and refractive index (RI) matching between tissue components. Such matching is obtained by partial replacement of interstitial water by a biocompatible agent that presents higher RI and it can be completely reversible by natural rehydration in vivo or by assisted rehydration in ex vivo tissues. Experimental data to characterize and discriminate between the two mechanisms and to find new ones are necessary. Using a simple method, based on collimated transmittance and thickness measurements made from muscle samples under treatment, we have estimated the diffusion properties of glucose, ethylene glycol (EG) and water that were used to perform such characterization and discrimination. Comparing these properties with data from literature that characterize their diffusion in water we have observed that muscle cell membrane permeability limits agent and water diffusion in the muscle. The same experimental data has allowed to calculate the optical clearing (OC) efficiency and make an interpretation of the internal changes that occurred in muscle during the treatments. The same methodology can now be used to perform similar studies with other agents and in other tissues in order to solve engineering problems at design of inexpensive and robust technologies for a considerable improvement of optical tomographic techniques with better contrast and in-depth imaging.

Concentration dependence of the optical clearing effect created in muscle immersed in glycerol and ethylene glycol

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 samples 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 solutions 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.

Skeletal muscle dispersion (400‐1000 nm) and kinetics at optical clearing

Skeletal muscle dispersion and optical clearing (OC) kinetics were studied experimentally to prove the existence of the refractive index (RI) matching mechanism of OC. Sample thickness and collimated transmittance spectra were measured during treatments with glucose (40%) and ethylene glycol (EG; 99%) solutions and used to obtain the time dependence of the RI of tissue fluids based on the proposed theoretical model. Calculated results demonstrated an increase of RI of tissue fluids and consequently proved the occurrence of the RI matching mechanism. The RI increase was observed for the wavelength range between 400 and 1000 nm and for the 2 probing molecules explored. We found that for 30 min treatment with 40% glucose and 99% EG, RI of sarcoplasm plus interstitial fluid was increased at 800 nm from 1.328 to 1.348 and from 1.328 to 1.369, respectively.

Simple multimodal optical technique for evaluation of free/bound water and dispersion of human liver tissue

Abstract. 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 8.2×10−7u2009u2009cm2/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 colorectal muscle in visible/NIR range

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.