Bertrand Beauvoit

Contribution of the mitochondrial compartment to the optical properties of the rat liver: a theoretical and practical approach.

The purpose of this work was to analyze the contribution of the mitochondria to the optical properties, i.e., light absorption and scattering, of the blood-free rat liver. Firstly, a theoretical model of the reduced scattering coefficient of the liver was performed by using the Mie theory, the Rayleigh-Debye-Gans approximation, and the electron microscopy descriptions of the liver ultrastructure. Compared with the hepatocyte volume, the nucleus and the peroxisomes, the mitochondria compartment, accounting for 22% of the liver cell volume, seemed to be the predominant factor for the light scattering of the liver. Second, by using time-resolved spectroscopy and a sample substitution method, we have measured the absorption and reduced scattering coefficients of blood-free perfused rat livers, isolated hepatocyte suspensions, and isolated mitochondria suspensions. A subsequent extrapolation of the isolated mitochondria data to the in vivo mitochondrial content and a comparison with the whole liver measurements lead to the following conclusions: 1) the mitochondria account for about 50% of the liver absorption coefficient at 780 nm (mu a = 0.25 cm-1 extrapolated from isolated mitochondria vs. 0.53 +/- 0.05 cm-1 measured for the liver); and 2) the mitochondrial compartment is the primary factor for the light scattering in the rat liver (mu s = 15.5 cm-1 extrapolated from the isolated mitochondria versus 15.9 +/- 2.4 cm-1 measured for the liver), demonstrating the relevancy of our preliminary theoretical study.

Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity

Additions of a solute/carbohydrate in tissue affect the size of tissue cells and the refractive indexes of the extra- and intracellular fluids, and thus the overall tissue scattering properties. We use both the Rayleigh- Gans and Mie theory approximation in calculating effects of the osmolarity and refractive indexes on the reduced scattering coefficient of tissue, and employ photon diffusion theory to associate the reduced scattering coefficient to the mean optical path length. The calculations show that changes of scattering in tissue depend not only on the change in extracellular refractive index but also on the change in osmolarity, and thus on the change in cell size and volume fraction. Experimentally, we have utilized time-domain and frequencydomain NIR techniques to measure the changes of optical properties caused by an addition of a solute in ntissue models and in perfused rat livers. The temperature-dependent path length measurement of the perfused liver confirms the dependence of tissue scattering on the tissue cell size. The results obtained from the liver with three kinds of carbohydrate perfusion display different scattering aspects and can be well explained by changes in cell size and in extracellular as well as intracellular refractive indexes. The consistency between the theoretical and experimental results confirms the dependence of optical properties in (liver) tissue on both tissue osmolarity and relative refractive indexes between the extracellular and intracellular compartments. This study suggests that the NIR technique is a novel and useful tool for noninvasive, physiological monitoring.

Tumor Localization Using Fluorescence of Indocyanine Green(ICG) In Rat Models

The permeability of tumor blood vessels to contrast agents (Gd chelates) has formed the basis for MRI breast tumor identification. It is also believed that angiogenesis starts before further tumor growth. Here we report tumor detection and localization using the fluorescence of Indocyanine Green (ICG). ICG fluorescence is excited by CW NIR laser light with a central wavelength of 830 nm is recored as a function of time. Differential fluorescence signals were observed after a low-dose intraveneous injection of ICG aqueous solution in rat model experiments. The difference between the fluorescence signal from the tumor side and the fluorescence signal from the control side is detectable even when the tumor is very small. During the tumor exponential growth phase, the ratio of these two signals is approximately 2.5; the ratio of the initial ICG clearance velocity in the tumor leg to that in the control leg is about 3. New investigations on human subjects with breast tumors, or reoccurrence after breast tumors were removed, are underway, and preliminary differential fluorescence signals have been observed.

Characterization of absorption and scattering properties for various yeast strains by time-resolved spectroscopy.

An understanding of the optical properties of biological media and cells is essential to the development of noninvasive optical studies of tissues. Unicellular organisms offer a unique opportunity to investigate the factors affecting light propagation, since they can be manipulated in ways impossible for more complex biological samples. In this study, we examined optical absorption and scattering properties of strongly multiple scattering yeast suspensions by means of near-infrared (NIR) time-resolved spectroscopy (TRS) and a sample substitution method. We determined the critical parameters for photon migration by varying the cell organelle content, the cell ploidy, the cell size, and the concentration of suspended cells. The results indicate that the photon absorption is insensitive to cell differentiation and that the cell volume is the primary factor determining light-scattering property.

Optical determination of fatty change of the graft liver with near-infrared time-resolved spectroscopy

A novel method for quantifying the fatty change of the graft liver by characterizing the optical property of the tissue was introduced. A wide range of lipid content in the rat liver was obtained by using different feeding regimens, with lipotropic chow (choline/methionine deficient or low chow). The liver was removed and flushed with Krebs-Ringer buffer solution with 3% albumin, and the optical properties of the liver, i.e., absorption and reduced scattering coefficients (mu(a) and mu(s)), were measured by time-resolved spectroscopy. The fatty liver showed lower mu(a) and higher mu(s) than the normal liver. Lower mu(a) and lower succinate dehydrogenase activity of the fatty liver suggested that the decrease in mu(a) might indicate a decrease in the mitochondrial content. The value of mu(s) was positively correlated with the lipid content of the liver, which indicates that fat droplets inside the hepatocyte act as dominant scatterers. To subtract the contribution of the mitochondrial compartment to mu(s), the ratio of mu(s) to mu(a) (mu(s):mu(a)) was useful for the assessment of the lipid content of the liver. These findings were also relevant with prediction of light scattering by the Mie theory. It was concluded that mu(a) and mu(s) of the graft liver, measured by time-resolved spectroscopy, can be useful parameters for quantifying the fatty change of the graft liver.

Application of near-infrared time-resolved spectroscopy to rat liver--a preliminary report for surgical application.

The applicability of near-infrared time-resolved spectroscopy to rat liver surgery was investigated. First, the technical reliability in determining the absorption coefficient (mu(a)) and reduced scattering coefficient (mu(s)) of the liver was checked. Next, boundary effects in determining mu(a) and mu(s) of the rat liver were examined. Finally, changes in mu(a) and mu(s) of rat liver with ischaemia were directly measured by TRS. Our TRS system showed that the mu(a) value held a linear correlation with the ink concentration in a lipid emulsion until mu(a) reached 1.2 cm(-1), while the mu(s) was fairly independent. The mu(a) values of blood-free rat liver and blood-containing rat liver at 780 nm were observed to be 0.43 cm(-1) and 0.67 cm(-1) by using the matching method, indicating that TRS is reliable in determining mu(a) and mu(s) of the liver. Possible errors in mu(a) and mu(s) determination due to the boundary effects of the rat liver were as small as 7%, when the mu(a) value was as high as observed for the liver. The oxygen saturation of haemoglobin (SO2) was changed from 64.9% to 8.0%, and the haemoglobin content (THB) from 189.1 microM to 131.6 microM by ischaemia. Mu(s) dynamically changed in the range 7.06 cm(-1) to 11.36 cm(-1). We conclude that time-resolved measurement is applicable in the high-mu(a) region observed in the liver, and can give quantitative estimations of SO2 and THB in the liver.

Time-resolved spectroscopy of mitochondria, cells, and rat tissues under normal and pathological conditions

In this study, the detailed dependence of the light scattering on the tissue architecture and intracellular composition was investigated. The reduced scattering coefficient ((mu) s) of isolated rat liver mitochondria, isolated liver cells and various rat tissues was measured at 780 nm by using time-resolved spectroscopy and a sample-substitution protocol. In a first part, extrapolations of the in vitro data to the in vivo situation showed that the mitochondrial compartment contributes for 73% of the scattering of the hepatocytes and about 100% of that of the whole liver. Finally, by analyzing different normal rat tissues and tumors, we have shown that the tissue (mu) s is independent on the cell concentration in the tissue but is roughly proportional to the tissue mitochondrial content.

Near-infrared spectroscopy of a heterogeneous turbid system containing distributed absorbers

In most biological tissues, absorbers such as blood in the blood vessels are localized within a low-absorbing background medium. To study the effect of distributed absorbers on the near infrared reflectance, we developed a Monte Carlo code and performed time-domain measurements on heterogeneous tissue-vessel models. The models were made of low absorbing polyester resin mixed with TiO2 as scatters. A series of tubes with diameters of 3.2 or 6.4 mm were made in the resin sample. The volume ratio of the tubes to the total sample is about 20%. During the measurement, these tubes were filled with turbid fluids with different absorption coefficients to simulate blood in various oxygenation states. We found that the apparent absorption coefficient of the resin/tube system, determined by using the diffusion equation fit, can be approximated by a volume-weighted sum of the absorption coefficients of the different absorbing components. This approximation has to be replaced by a more complex expression if the difference in absorption between the absorbers and background is very large (approximately 20 times). The results of the tissue phantom study are supported by the Monte Carlo simulation. Possible explanations for the photon migration in this kind of heterogeneous system is also presented.

Changes in the light path length of blood-perfused rat liver by increased hematocrit and anoxia

The purpose of this study is to evaluate the usefulness of time-resolved spectroscopy (TRS) and phase modulation spectroscopy (PMS) for the measurement of hemoglobin saturation (SO2) in the liver. Our materials and methods were: (1) Absorption coefficient ((mu) a) and reduced scattering coefficient (microsecond(s) ) of in situ rat liver, blood-free rat liver and red blood cell (RBC) suspension were measured by TRS. (2) Changes in the light path length of blood-perfused rat liver by increasing hematocrit (3%, 12%, and 36%) and anoxia were measured by PMS. Our results show: (1) (mu) a of in situ rat liver could not be determined, because the absorption was too high. From (mu) a and microsecond(s) values of blood-free liver and RBC suspension, (mu) a and microsecond(s) of normal-hematocrit liver was extrapolated as 1.08 cm-1 and 15.46 cm-1 at 780 nm, respectively. (2) Although the trend of liver SO2 by increasing hematocrit was reasonable, there was a discrepancy between liver SO2 and output SO2 by anoxia. Changes in the light path length of the liver were as small as 10% of the total light path length. We conclude: Quantitation of liver SO2 by TRS and PMS was difficult because of its very high (mu) a. Since changes in the light path length were small, continuous wave spectroscopy would be an effective way to monitor the oxygenation state of the liver.

Determination of the blood oxygenation in the brain by time-resolved reflectance spectroscopy: contribution of vascular absorption and tissue background absorption

The possibility of measuring the blood oxygenation in the brain with near infrared light has been studied. The goal of this study was to quantify the influence of different brain layers on brain blood oxygenation measurements. Experimental results obtained from time resolved reflectance measurements on layered tissue phantoms were compared to Monte Carlo simulations of layered models, diffusion theory, and in vivo measurements on the human head. Both the experimental results and simulations show that the absorption coefficient (mu) a, which is closely related to the blood oxygenation, of deeper layers can be accessed in the time domain. Thus fitting analytical expressions found from diffusion theory only to the late part of the time resolved reflectance allows us to determine (mu) a and subsequently the blood oxygenation of the deepest medium (e.g. brain tissue).