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.

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title: Time resolved reflectance and transmittance for the non-invasive measurement of tissue optical properties.
abstract: When a picosecond light pulse is incident on biological tissue, the temporal characteristics of the light backscattered from, or transmitted through, the sample carry information about the optical absorption and scattering coefficients of the tissue. We develop a simple model, based on the diffusion approximation to radiative transfer theory, which yields analytic expressions for the pulse shape in terms of the interaction coefficients of a homogeneous slab. The model predictions are in good agreement with the results of preliminary in vivo experiments and Monte Carlo simulations.

title: Time-dependent optical spectroscopy and imaging for biomedical applications : Lasers in medicine
abstract: While optical spectroscopy and imaging are essential tools in science and engineering, their application in living tissue is complicated by multiple scattering of light. In spectroscopy, this scattering causes uncertainty in the pathlength traveled by photons in the tissue, while images suffer reduced resolution and contrast. Picosecond light sources and fast detectors have made it possible to address these problems by direct measurement of the photon time-of-flight

title: Comparison of time-resolved and -unresolved measurements of deoxyhemoglobin in brain.
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title: Quantitation of time- and frequency-resolved optical spectra for the determination of tissue oxygenation.
abstract: The recent development of near-infrared time- and frequency-resolved tissue spectroscopy techniques to probe tissue oxygenation and tissue oxygenation kinetics has led to the need for further quantitation of spectroscopic signals. In this paper, we briefly review the theory of light transport in strongly scattering media as monitored in the time and frequency domains, and use this theory to develop algorithms for quantitation of hemoglobin saturation from the photon decay rate (delta log R/delta t) obtained using time-resolved spectroscopy, and from the phase-shift (theta) obtained from frequency-resolved, phase-modulated spectroscopy. To test the relationship of these optical parameters, we studied the behavior of delta log R/delta t and theta as a function of oxygenation in model systems which mimicked the optical properties of tissue. Our results show that deoxygenation at varying hemoglobin concentrations can be monitored with the change in the photon decay kinetics, delta delta log R/delta t in the time-resolved measurements, and with the change in phase-shift, delta theta, in the frequency-resolved technique. Optical spectra of the adult human brain obtained with these two techniques show similar characteristics identified from the model systems.

title: Light Scattering by Small Particles
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title: Wave propagation and scattering in random media
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title: Absorption and Scattering of Light by Small Particles
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title: Angular dependence of scaftering from Escherichia coli cells.
abstract: The scattering of visible light from randomly oriented systems of heterogeneous E. coli cells in water was measured as a function of angle for 10-90 degrees . The cells are assumed to be prolate ellipsoids of revolution whose dimensions are independently measured. For these cultures the average cell volume and axial ratio is 0.6 micro(3) and 2.81, respectively. Using the measured dimensions, the Rayleigh-Debye approximation for the homogeneous ellipsoid was used to calculate theoretical scattering. A newly developed Rayleigh-Debye equation for a coated ellipsoid was also used. Both cell models lead to good theoryexperiment agreement, especially the coated ellipsoid model. The absolute position of the two observed scattering minima and the wavelength dependence of these positions is in excellent agreement with the theoretical predictions. The existence of the scattering minima in randomly oriented heterogeneous cultures of E. coli is attributed to the uniformity of their minor axes.

title: Osmotic response of lymphocytes measured by means of forward light scattering: theoretical considerations.
abstract: Recent data provide solid experimental evidence that increase in cell size of human lymphocytes, caused by osmotic stress, is not directly proportional to forward scattering intensity but rather is inversely proportional. Here, we provide arguments that this phenomenon can be quantitatively explained by means of a model based on a modified Rayleigh-Debye-Gans theory. As a consequence, the current view that forward scattering intensities can be used as a measure of gross cell size needs to be reconsidered. In addition, we postulate that structural changes of the cytoplasm, imposed by an osmotic stress, reflect corresponding changes in the nucleus.

title: Comparison of Mie theory and the light scattering of red blood cells.
abstract: Two important optical properties of red blood cells (RBCs), their microscopic scattering cross sections sigma(s), and the mean cosine of their scattering angles micro, contribute to the optical behavior of whole blood. Therefore, the ability of Mie theory to predict values of sigma(s) and was tested by experiment. In addition, the effect of red blood cell size on sigma(s) and micro was investigated in two ways: (1) by studying erythrocytes from the dog, goat, and human, three species known to have different RBC sizes and (2) by allowing the RBCs from each species to shrink or swell osmotically. Values of sigma(s) obtained by measuring the collimated transmittance of dilute RBC suspensions illuminated with a He-Ne laser agreed with those predicted by Mie theory. Moreover, measured as values were directly proportional to RBC volume. By contrast, values of from Mie theory were consistently greater than those obtained experimentally by making angular scattering measurements in a goniometer. Thus Mie theory appears to yield adequate values for the RBC's microscopic scattering cross section, but by treating the RBC as a sphere with an equal volume, Mie theory fails to take the RBC's anisotropy into account and thus yields spuriously high values for micro.

title: Measurements of angular distributions of Rayleigh and Mie scattering events in biological models.
abstract: Remission spectroscopy in living tissues can only be performed because scattering processes induce a pronounced amount of backscattered light. New types of scattering chambers were constructed in order to investigate the angular distribution of light intensities caused by scattering events typical for living tissues. Different solutions and suspensions containing microspheres, haemoglobin molecules, erythrocytes and liver homogenate were used in order to gain basic information applicable to remission spectrophotometry in tissue. Reflection spectroscopy in biological tissues is only possible because of the scattering properties of the material under observation. Light which penetrates the sample is remitted from the tissue. The remission is caused by the scattering of the light by different biological structures and particles. This scattered light gives information about the optical properties of the particles and structures in an integral form. A scattering chamber for small sample volumes has been developed for measurements of the optical properties of various particles in suspension. Measurements of scattering diagrams from microspheres with an average diameter of 7 microns, artificial particles, haemoglobin solutions, mixtures of microspheres and haemoglobin as well as erythrocytes and mitochondria have been performed. The different optical behaviour of the measured signals is qualitatively discussed.

title: Mitochondrion of Yeast: Ultrastructural Evidence for One Giant, Branched Organelle per Cell
abstract: Three-dimensional models constructed from 80 to 150 consecutive serial sections of entire yeast cells showed that all the separate mitochondrial profiles were cross sections through a single, branching, tubular structure about 50 to 60 micrometers in length and 200 to 600 nanometers in diameter. The data are contrary to conventional notions of mitochondrial size, form, and number per cell and should lead to a reassessment of mitochondrial genetics and biogenesis.

title: Fluorescence microscopic studies of mitochondrial nucleoids during meiosis and sporulation in the yeast, Saccharomyces cerevisiae.
abstract: Configurational changes of mitochondria and mitochondrial nucleoids (mt-nucleoids) during meiosis and sporulation in the yeast, Saccharomyces cerevisiae, were examined using the mitochondrial membrane-binding fluorescent dye, dimethyl aminostyrylmethylpyridiniumiodine (DASPMI) and the DNA-binding fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI). In zygotes just after mating, mt-nucleoids were observed as many small discrete light spots in the cytoplasm. During meiosis in zygotes, mt-nucleoids at first coalesced with each other into a long string and then separated into spherical nucleoids in four spores. These changes paralleled those in mitochondria observed using DASPMI. The use of spheroplasts allowed us to examine the behaviour of mt-nucleoids at higher resolution and to identify several distinct meiotic prophase stages of the cell nucleus during early sporulation. In diploid spheroplasts at the stationary phase, 50-70 of the mt-nucleoids were observed to be separated from each other and each spherical mitochondrion contained only one mt-nucleoid. At the later stage of premeiotic DNA synthesis, a single branched giant mitochondrion was formed as a result of complete mitochondrial fusion. All of the mt-nucleoids were arranged in an array on a giant mitochondrion and coalesced into a string-like network. Through meiosis I and II, strings of mt-nucleoids were observed close to the dividing nuclei. At late meiosis II, a ring of mt-nucleoids enclosing each daughter nucleus was formed. In ascospores, discrete small nucleoids were visible close to each spore nucleus with a 'string-of-beads' appearance. Many mt-nucleoids were excluded from the ascospores and remained in the residual cytoplasm of the ascus.

title: Localization of mitochondria in living cells with rhodamine 123.
abstract: The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.

title: Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.
abstract: By using a potential-dependent J-aggregate-forming delocalized lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1), we find that membrane potentials across mitochondria in a living cell can be heterogeneous. Remarkably, even within a long contiguous mitochondrion, regional heterogeneity in membrane potentials appears to be possible.

title: Hormone-induced intracellular calcium oscillations and mitochondrial energy supply in single hepatocytes.
abstract: Mitochondria in liver cells behave as a regulated stable power supply — optimized with respect to efficiency and stability against fluctuations of ATP-utilizing reactions in the case of starvation — even in the presence of intracellular Ca 2+ oscillations.

title: Three‐dimensional ultrastructure of normal rat hepatocytes by quick‐freezing and deep‐etching method
abstract: The three‐dimensional ultrastructure of nuclei and cell organelles including rough‐surfaced endoplasmic reticulum (RER), mitochondria, and cytoskeleton were studied in normal rat hepatocytes by the quick‐freezing and deep‐etching method. Peroxisomes and mitochondria were observed as spherical structures with granular matrices. Peroxisomes were identified by their size and matrices, which were more condensed than those of mitochondria. Ribosomes were identified as granular structures and were attached to the surface of endoplasmic reticulum. Cytoskeletal filaments were identified by their differences in diameter on the replica membranes, as well as in conventional ultrathin sections. Microfilaments were mainly localized around the bile canaliculi and adjacent to sinusoids. Intermediate filaments were observed around the bile canalicular microfilaments. Only a few filaments were observed near the lateral plasma membranes. Cross‐bridges measuring 5–7 nm in diameter were localized between the lamellae of RER and the surface of mitochondria. The quick‐freezing and deep‐etching method could be used to clarify the three‐dimensional association between the cytoskeleton and membrane‐bound organelles in hepatocytes.

title: Studies on Giant Mitochondria
abstract: Mitochondria, which play a cardinal role in the economy of the cell, are excellent morphological indicators of the state of health of cells. One of the ways in which these organelles respond to metabolic injury is to increase in size, often to a point where they merit the appellation of giant mitochondria or megamitochondria. Although quite large, even gigantic, mitochondria may occur in certain normal cells,’ it is those organelles that have become greatly enlarged as a result of pathological processes in typical cells such as hepatocytes or cardiomyocytes that are of concern in this review. Giant mitochondria have been reported to occur in variety of human diseases and in liver cells of aged humans2-I6 (TABLE 1). Such mitochondria are usually observed retrospectively in fixed specimens and, because of their source, rarely are available for experimentation. Obviously, giant mitochondria that are experimentally induced in laboratory animals are indispensable to the study of altered mitochondria1 morphology and function. Giant mitochondria have been experimentally produced in rodents, principally in their hepatocytes (but in other animals and cell types as well), by nutritional m a n i p u l a t i ~ n ’ ~ ~ ~ ~ (TABLE 2), pharmacological agents2949 (TABLE 3), ethanol intoxication5a4 (TABLE 4), and Giant mitochondria in liver cells have one of three basic morphologies, depending on the means whereby they are induced. Hepatic megamitochondria occurring in mice fed a riboflavin-deficient diet may be up to 10 pm in diameter” (FIG. 1). They tend to be spherical and are very similar to, although considerably larger than, normal-sized mitochondria in the same cells. Within a unit cross-section area, the number and distribution of cristae are the same regardless of organelle size, although some megamitochondria may contain a stack of cristae. Intramitochondrial dense granules are scattered throughout the matrix of the giant mitochondria, which is of normal density (FIG. 2). I n contrast, giant hepatic mitochondria (greater than 10 pm in diameter) resulting from feeding mice or rats r~ialarnide;~ ethidium or the copper-chelating agent, c ~ p r i z o n e * ~ ~ ~ (FIG. 3), or from injection with triamcinolone,68 have short cristae that are restricted to the organelle periphery (FIG. 4); the matrix, which is greatly expanded, is of near normal density but lacks membranes. It should be noted that these seemingly diametrically opposed arrangements of cristae are not invariably related to a particular method of megamitochondrial induction. When galactoflavin, a reversible antagonist of riboflavin, is added to a riboflavin-deficient diet, the appearance of symptoms of ariboflavinosis is greatly accelerated, with giant mitochondria being observed in mice as early as 13 days after initiation of the diet, (TABLE 5).

title: A QUANTITATIVE STEREOLOGICAL DESCRIPTION OF THE ULTRASTRUCTURE OF NORMAL RAT LIVER PARENCHYMAL CELLS
abstract: The principles of stereology have been applied to a morphometric analysis of parenchymal cells from the peripheral, midzonal, and central regions of normal rat liver lobules. The fractional volumes of cytoplasm occupied by mitochondria, peroxisomes, lysosomes, lipid, and glycogen have been determined. The surface densities of smooth- and rough-surfaced endoplasmic reticulum and of mitochondrial envelope and cristae have also been measured. The average number and dimensions of mitochondria and peroxisomes have been evaluated. By the use of an independent measurement of the average cytoplasmic volume, these data have been expressed as the actual volumes, areas, and numbers per cell in the different parts of the hepatic lobule. Similarly, the volumes of the envelope, cristae, and matrix compartments and the area of cristae membranes have been calculated for the average-sized mitochondrion in each lobular zone. Structural homogeneity is found in over 80% of normal rat liver parenchymal cells, with most of the significant differences being confined to those cells immediately surrounding the central veins.

title: Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study
abstract: When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat- storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.

title: Biochemical and stereological analysis of rat liver mitochondria in different thyroid states
abstract: The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.

title: Structural reaction pattern of hepatocytes following exposure to hypotonicity
abstract: Isolated rat hepatocytes were exposed to hypotonic media (225 mosmol/l) for 5 and 15 min and processed for a quantitative electron microscopic stereologic analysis. Within 5 min of hypotonicity, the hepatocyte volume increased by 25% and thereatter displayed a volume regulatory decrease leading to mean cellular volume, which was 16% above that of controls. Stereologic analysis of the major subcellular compartment, the cytosol, showed an identical change as the whole cell. In contrast to that, the mitochondrial compartment increased in volume by 30% within the first 5 min of exposure and returned by regulatory volume decrease back to values of the isotonic controls after 15 min of hypotonicity. In contrast, hypotonicity (220 mosmol/l)‐stimulation of flux through mitochondrial glutaminase and the glycine cleavage enzyme complex, as assessed by 14CO2 production from [1‐14C]glutamine or [1‐14C]glycine in isolated perfused rat liver persisted throughout a 15‐min period of hypotonic exposure. Thus hypotonicity‐induced alterations of mitochondrial metabolism apparently do not parallel the time course of mitochondrial volume changes. This suggests that persistent mitochondrial swelling is not required for functional alterations, but that the latter may be triggered by the initial swelling of mitochondria. Hypotonic exposure did not alter the nuclear volume of isolated hepatocytes. Cell membrane surface nearly doubled after 5 min of hypotonic exposure, but returned within 15 min of exposure to values observed in normotonic media. This may reflect the participation of exocytosis in hepatocyte volume regulation.

title: The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism.
abstract: The purpose of this article is to describe briefly the methods by which the intra-mitochondrial volume may be measured both in vitro and in situ, to summarise the mechanisms thought to regulate the mitochondrial volume and then to review in more detail the evidence that changes in the intra-mitochondrial volume play an important part in the regulation of liver mitochondrial metabolism by glucogenic hormones such as glucagon, adrenaline and vasopressin. It will be shown that these hormones cause an increase in matrix volume sufficient to produce significant activation of fatty acid oxidation, respiration and ATP production, pyruvate carboxylation, citrulline synthesis and glutamine hydrolysis. These are all processes activated by such hormones in vivo. I will go on to demonstrate that the increase in matrix volume is brought about by an increase in mitochondrial [PPi]. This is able to stimulate K+ entry into the matrix, perhaps through an interaction with the adenine nucleotide translocase. The rise in matrix [PPi] is a consequence of an increase in cytosolic and hence mitochondrial [Ca2+] which inhibits mitochondrial pyrophosphatase. In the final section of the review I provide evidence that changes in mitochondrial volume may be important in the responses of a variety of tissues to hormones and other stimuli. I write as a metabolist with a working knowledge of bioenergetics rather than the converse, and this will certainly be reflected in the approach taken. If I cause offence to any dedicated experts in the field of bioenergetic by my ignorance or lack of understanding of their studies I can only offer my apologies and ask to be corrected.

title: Isolated Hepatocytes: Preparation, Properties and Applications
abstract: Chapter 1.  1.1 The development of the isolated hepatocyte preparation. 1.2 Factors involved in cellular organization and adhesion. 1.3 Mechanical methods for separation of parenchymal liver cells. 1.4 Development of enzymatic methods for isolated hepatocyte preparation.  Principles of cell separation. 1.6 Separation of cells from solid organs other than liver. 1.7 Advantages of the isolated hepatocyte preparation. Chapter 2. High-yield preparation of isolated hepatocytes from rat liver.   One-step method. 2.3 Two-step procedure.  Evaluation of common modifications to the recommended method. 2.5 Initial determination of cell quality. 2.6 Assessment of cell yield. 2.7 Reasons for unsatisfactory preparations. 2.8 Removal of damaged cells from the preparation 2.9 Storage of hepatocytes. Chapter 3. Other methods for hypatocyte preparation.  3.2 Isolated hepatocyte preparation from liver slices. 3.3 Preparation of intact isolated hepatocytes without collagenase. 3.4 Preparation of isolated hepatocytes from species other than rat. 3.5 Preparation of hepatocytes from foetal or neonatal animals. 3.6 Preparation of hepatocytes from abnormal animals. 3.7 Preparation of suspensions of damaged hepatocytes. 3.8 Preparation and purification of non-parenchymal cells. Chapter 4. Assessment of integrity of isolated hepatocytes.  4.2 Measures of cellular integrity. 4.3 Additional methods for determining cellular integrity. Chapter 5. Microscopy of isolated hepatocytes. 5.1 Light microscopy of isolated hepatocytes. 5.2 Morphology of isolated hepatocytes by transmission electron microscopy. 5.3 Techniques. 5.4 Morphological studies yet to be exploited. 5.5 Summary of studies by transmission electron microscopy. 5.6 Scanning electron microscopy. Chapter 6. Biochemical properties. 6.1 Basis for expressing cell activity. 6.2 Measurement of cellular composition. 6.3 Cellular composition 6.4 Experimental conditions. 6.5 Measurement of cellular O 2 -uptake. 6.6 Metabolic activities. 6.7 Transport activities. Chapter 7. Utilization of hepatocytes for drug studies.  7.2 Drug uptake and metabolism by isolated hepatocytes. 7.3 Isolated hepatocytes and drug toxicity. Chapter 8. Study the effects of hormones. 8.1 Optimal conditions for observing the effects of hormones. 8.3 Special conditions for the study of specific hormonal effects. 8.4 Action of insulin. 8.5 Action of steroid hormones. 8.6 Hormone effects on mitochondria isolated from hepatocytes. Chapter 9. Ca 2+ ion transport and compartmentation. 9.1 Measurement of intracellular Ca 2+ . 9.2 Changes in Ca 2+ movements monitored using 45 Ca 2+ . 9.3 Measurement of intracellular free Ca 2+ using fluorescent indicators. 9.4 Determination of rates of Ca 2+ inflow using glycogen phosphorylase or fluorescent Ca 2+ indicators. 9.5 Assessment of available techniques for the measurement of Ca 2+ in hepatocytes. Chapter 10.

title: [15] Isolation of liver or kidney mitochondria
abstract: Publisher Summary This chapter describes the isolation of liver or kidney mitochondria. The selected tissue is disrupted by homogenization in cold isotonic sucrose. Differential centrifugation is then employed to separate the mitochondria from cell debris, red blood cells, nuclei, microsomes, and soluble components. For the isolation of liver mitochondria, the homogenate is distributed into Lusteroid centrifuge cups and centrifuged at 600 g for 10 minutes. The supernatant fraction is decanted and saved. The pellets may be dispersed by using the side of a stirring rod against the wall of the cup or by handoperating the homogenizer. The resuspended material is centrifuged at 600 g for 10 minutes. The supernatant fractions are combined. The pellets are discarded. This washing contributes not only to the yield of the final mitochondrial preparation, but also to its integrity, apparently by permitting the recovery of the larger mitochondria. For the isolation of kidney, the kidney capsule is removed by gently squeezing the kidney through the thumb and forefinger. The kidney is then cut sagittally. The medullary portion is removed and discarded. Mitochondria are then prepared from the cortex following the method described for liver, with the exception that the mitochondrial pellet need be washed only once. Method for testing the quality of mitochondria is also discusses in the chapter.

title: Characterization of absorption and scattering properties of small-volume biological samples using time-resolved spectroscopy.
abstract: With time-resolved spectroscopy, we develop an experimental approach by sample substitution to measure the absorption (mu a) and reduced scattering (mu's) coefficients of small-volume biological samples. To investigate the method, small-volume control samples are substituted into a large-size host medium during increases in the absorber (or scatterer) concentration of the host. By characterizing the deviation of the spectra taken with and without the sample, we determine the matching points where the sample and surrounding medium are optically identical. We show that this method can result in correct values of the mu a and mu's for the sample within 6% error if the matching conditions for both the mu a and mu's are fully realized. The results also indicate that this method can give approximate values of the mu a and mu's in a reasonable range if either the mu a or the mu's matching between the two media is realized. This method has been applied to the studies of absorption properties of a human finger and of scattering properties of yeast.

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

title: Refractive index of some mammalian tissues using a fiber optic cladding method.
abstract: The index of refraction n of the many mammalian tissues is an important but somewhat neglected optical constant. Archival and oral papers have quoted the use of values of n for tissue generally ranging from 1.35 to 1.55. However, these values are frequently without experimental basis. They have arbitrarily used values near that of water, which is a major component of mammalian tissue, or have calculated a theoretical n from the weighted elemental composition of tissue. Since these values have not been precise and little information is available on specific indices for each tissue, a study was undertaken to develop a simple, rapid, and reliable method for the experimental determination of n. This was done using the ubiquitous quartz optical fiber. By substituting the usual cladding found on commercial quartz optics by the tissue in question and utilizing the principle of internal reflection, the value of n for the specific tissue can be calculated. This is done by utilizing the known indices for air and quartz and measuring the angle of the emergent cone of light from the output of the optical fiber. A number of indices for mammalian tissue (bovine, porcine, canine, and human) have been determined at 632.8 nm. With few exceptions, for tissues at this wavelength, n was in the 1.38-1.41 range. The species type did not appear to be a factor. Bovine muscle showed normal dispersion characteristics through the visible wavelengths. The denaturation of tissue was shown to alter significantly the refractive index.

title: Dynamic light scattering from polydisperse suspensions of large spheres. Characterization of isolated secretory granules.
abstract: Dynamic light scattering is useful in determining the diameter of submicrometer particles in suspension. When both static scattering intensity P(K) and apparent diffusion coefficient D can be measured in a wide range of the length of the scattering vector K, it is possible to determine the number-average diameter dn and sharpness in size distribution of spheres. We derived approximate, but very simple, expressions for mean value of P(K) and mean value of D/D(dn) applicable to very large spheres for which the so-called Rayleigh-Debye condition is perturbed, where mean value of ... stands for the size average. These approximate expressions were compared with the numerical results based on the Mie scattering theory. Experimental results for isolated secretory granules, zymogen (dn approximately 800 nm) and chromaffin (dn approximately 400 nm) granules, were analyzed by use of the present formulation, and the dispersion in size distribution, sigma/dn = [(the mean of d2)/d2n - 1]1/2, was found to be about 0.2 for both types of granules.

title: DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION
abstract: Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ3 particle volume The mean volume of rat liver mitochondria was 0.42 µ3 or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 103, and per gram of rat liver was about 11 x 1010. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ3 and from rat kidney cortex, 0.23 µ3. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a 3), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement

title: Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study
abstract: The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.

title: Integrated stereological and biochemical studies of hepatocytic membranes. I. Membrane recoveries in subcellular fractions
abstract: Previous attempts to relate the structure and function of hepatocytic membranes have compared biochemical data of fractions to morphological data derived from either intact tissue or fractions. The effects of the original homogenization aside, biochemical recoveries comparing membrane marker enzymes of the homogenate to subsequent fractions suggest a general conservation of activity. A sterological study was undertaken to estimate membrane surface areas in the intact tissue, homogenate, and fractions of the same livers and then to test the comparability of these data with membrane marker enzymes by calculating both morphological and biochemical recoveries. The sterological data were corrected for errors due to section thickness and compression. The average total membrane sufrace area per 1 g of liver was 9.3 m2 in the intact tissue (T), 7.8 m2 in the homogenate (H), and 7.4 m2 in the fractions (F); recoveries for the membrane surface areas thus averaged 96% for the (F/H) and 81% for the (F/T) comparisons. In homogenate and fractions, the differentiability of membranes by morphological criteria was limited to rough- and smooth- surfaced membranes, as well as outer and inner mitochondrial membranes. The recoveries of rough-surfaced membranes were 101% for F/H and 92% for F/T; those of smooth-surface membranes were 89% for F/H and 107% for F/T. For mitochondrial membranes, a recovery of 100% for F/H was obtained, whereas it amounted to only 54% for F/T. With respect to F/H, the membrane recoveries compare well with the marker enzyme recoveries obtained biochemically. The extension of recovery calculations to the intact tissue (F/T) revealed satisfactory conservation of the procedures of homogenization and fractionation; it indicates, however, that a shift of a substantial part of mitochondrial membranes to the pool of unidentifiable smooth membranes may occur on homogenization.

title: Hemoglobin as the red pigment of microsomes.
abstract: The red color of isolated microsomes results from the presence of adsorbed hemoglobin. Adsorption occurs only at very low ionic strengths. In contrast to microsomes, mitochondria do not adsorb hemoglobin. Hemoglobin added to tissue homogenates is useful as a specific stain to differentiate microsomal from mitochondrial material in mixed centrifuge pellets.

title: Hemoperfusion, rate of oxygen consumption and redox levels of mitochondrial cytochrome c (+c1) in liver in situ of anesthetized rat measured by reflectance spectrophotometry.
abstract: Utilizing reflectance spectrophotometry, hemoperfusion, rate of oxygen consumption and redox level of mitochondrial cytochrome c (+c1) in livers in situ of anesthetized rats were measured. The transition to the anoxic state was induced by raising the pressure on the liver surface to more than the hepatic blood pressure by pressing with the tip of the optical guide of the reflectance spectrophotometer. During this transition, the average oxygen saturation of hemoglobin in the liver in situ decreased linearly with time until it became 10--20% of the total. This was followed by reduction of mitochondrial cytochrome c (+c1), which reached completion in 10--20 s. The measured O2 consumption rate remained constant until the percentage of oxyhemoglobin in situ decreased to a critical level. There was then a decrease in the rate of O2 consumption which was accompanied by a progressive reduction of cytochrome c (+c1). It was shown that amounts of hemoglobin and mitochondrial respiratory chain cytochromes in the liver in situ could be measured non-invasively and could provide important signals for vital cellular functions. The changes in hemoperfusion and rate of O2 consumption of the liver in situ following ethanol ingestion were also shown in rats, and are briefly discussed with respect to possible application of this method to study the pathophysiology of tissues.

title: The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia.
abstract: The resolution of cytochrome and hemoglobin changes in in vivo rat and cat brains has required studies over wide wavelength ranges (580-1100 nm) with a novel spectroscopic technique using blood-free and blood-perfused brains. Tissue oxygen was varied from physiological levels to 0 and hematocrits were varied from normal to less than 1%. The experimental results were subjected to a multicomponent analysis using the Beer-Lambert law. At normal hematocrits, the oxygen saturation of hemoglobin in the brain was found to be 30-50% in rats and cats, indicating that the optical method responded primarily to the saturation of the venous ends of the capillary beds. With low hematocrits, both brains showed the absorption band of reduced cytochrome c, the iron component of cytochrome aa3, plus the absorption band of the oxidized copper component. In cat brains, the background absorption changed at all wavelengths. Thus, no isosbestic points were observed in the spectra. In rat brains, however, they were readily observed. The "overtones" of water absorption in the NIR region were found to be significant in the difference spectra of the cat brain, but not in the rat brain. Parallel absorbance changes in the heme and copper components of cytochrome aa3 were obtained in rat and cat brains during the normoxic-hypoxic transition. The ratio of the iron absorbance at 605 nm to the copper absorbance at 830 nm is much smaller in both brains than the in vitro value due to the shorter path length of photon migration at the shorter wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)