The Experts below are selected from a list of 1902 Experts worldwide ranked by ideXlab platform
Pontus Aspenstrom - One of the best experts on this subject based on the ideXlab platform.
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RhoD localization and function is dependent on its gtp gdp bound state and unique n terminal motif
European Journal of Cell Biology, 2018Co-Authors: Magdalena Blom, Katarina Reis, Pontus AspenstromAbstract:The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics.
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the atypical rho gtpase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
Experimental Cell Research, 2017Co-Authors: Magdalena Blom, Katarina Reis, Johan Heldin, Johan Kreuger, Pontus AspenstromAbstract:RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.
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RhoD is a golgi component with a role in anterograde protein transport from the er to the plasma membrane
Experimental Cell Research, 2015Co-Authors: Magdalena Blom, Vishal Nehru, Annica K B Gad, Katarina Reis, Hans Blom, Pontus AspenstromAbstract:RhoD is a member of the Rho GTPase family and it coordinates actin dynamics and membrane trafficking. Activation of RhoD results in formation of filopodia, dissolution of stress fibers, and the subsequent formation of short actin bundles. In addition, RhoD localizes to early endosomes and recycling endosomes, and has a regulatory role in endosome trafficking. In this study, we report on a function of RhoD in the regulation of Golgi homeostasis. We show that manipulation of protein and activation levels of RhoD, as well as of its binding partner WHAMM, result in derailed localization of Golgi stacks. Moreover, vesicle trafficking from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus measured by the VSV-G protein is severely hampered by manipulation of RhoD or WHAMM. In summary, our studies demonstrate a novel role for this member of the Rho GTPases in the regulation of Golgi function.
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atypical rho gtpases RhoD and rif integrate cytoskeletal dynamics and membrane trafficking
Biological Chemistry, 2014Co-Authors: Pontus AspenstromAbstract:The Rho GTPases are essential regulators of basic cellular processes, including cell migration, cell contraction and cell division. Most studies still involve just the three canonical members, RhoA, Rac1 and Cdc42, although the Rho GTPases comprise at least 20 members. The aim of this review is to highlight some of the recent advances in our knowledge regarding the less-studied Rho members, with the focus on RhoD and Rif. The phenotypic alterations to cell behaviour that are triggered by RhoD and Rif suggest that they have unique impacts on cytoskeletal dynamics that distinguish them from the well-studied members of the Rho GTPases. In addition, RhoD has a role in the regulation of intracellular transport of vesicles. Taken together, the available data indicate that RhoD and Rif have functions as master regulators in the integration of cytoskeletal reorganisation and membrane trafficking.
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RhoD binds the rab5 effector rabankyrin 5 and has a role in trafficking of the platelet derived growth factor receptor
Traffic, 2013Co-Authors: Vishal Nehru, Oleksandr Voytyuk, Johan Lennartsson, Pontus AspenstromAbstract:RhoD is a member of the classical Rho GTPases and it has essential roles in the regulation of actin dynamics. RhoD localizes to early endosomes and recycling endosomes, which indicates its important role in the regulation of endosome trafficking. Here, we show that RhoD binds to the Rab5 effector Rabankyrin-5, and RhoD and Rabankyrin-5 colocalize to Rab5-positive endosomes, which suggests a role for Rabankyrin-5 in the coordination of RhoD and Rab5 in endosomal trafficking. Interestingly, depletion of RhoD using siRNA techniques interfered with the internalization of the PDGFβ receptor and the subsequent activation of the downstream signaling cascades. Our data suggest that RhoD and Rabankyrin-5 have important roles in coordinating RhoD and Rab activities during internalization and trafficking of activated tyrosine kinase receptors.
Anne J Ridley - One of the best experts on this subject based on the ideXlab platform.
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rhoa rhob and rhoc have different roles in cancer cell migration
Journal of Microscopy, 2013Co-Authors: Anne J RidleyAbstract:Rho GTPases are well known to regulate cell motility through activation of a variety of downstream effector proteins, including enzymes, adaptor proteins and actin nucleators. The three closely related Rho GTPases RhoA, RhoB and RhoC all have the potential to interact with the same downstream effectors, yet they have substantially different effects on cell shape and migratory properties. Here I review the different ways in which RhoA, RhoB and RhoC expression is regulated in cancer and how they play distinct roles in cancer progression. I describe their main effectors known to contribute to cell motility. Recent results from our laboratory and others indicate that RhoA, RhoB and RhoC can be activated by specific stimuli and act through different effectors to control distinct aspects of cancer cell migration and invasion. This suggests that they each make unique contributions to cancer by participating in different protein complexes.
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why three rho proteins rhoa rhob rhoc and cell motility
Experimental Cell Research, 2004Co-Authors: Ann P Wheeler, Anne J RidleyAbstract:Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.
John J. Lemasters - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial calcium transients in adult rabbit cardiac myocytes inhibition by ruthenium red and artifacts caused by lysosomal loading of ca2 indicating fluorophores
Biophysical Journal, 2000Co-Authors: Donna R. Trollinger, Wayne E. Cascio, John J. LemastersAbstract:A cold/warm loading protocol was used to ester-load RhoD 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial RhoD 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial RhoD 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.
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Confocal Imaging of Both Mitochondrial and Cytosolic Free Ca2+ in Cardiac Myocytes Co-Loaded with RhoD 2 and Fluo 3: Inhibition by Ruthenium Red of Mitochondrial but not Cytosolic Ca2+ Transients
Microscopy and Microanalysis, 1998Co-Authors: Donna R. Trollinger, Wayne E. Cascio, John J. LemastersAbstract:Previously, we showed rapid mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake by the Ca2+ uniporter, inhibits mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca2+ transients.Ca 2+-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM RhoD 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial RhoD 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of RhoD 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.
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selective loading of RhoD 2 into mitochondria shows mitochondrial ca2 transients during the contractile cycle in adult rabbit cardiac myocytes
Biochemical and Biophysical Research Communications, 1997Co-Authors: Donna R. Trollinger, Wayne E. Cascio, John J. LemastersAbstract:A strategy of cold loading of the Ca2+-indicating fluorophore RhoD 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial RhoD 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of RhoD 2. These experiments directly demonstrate rapid mitochondrial free Ca2+ transients during the contractile cycle in rabbit cardiac myocytes.
Daniel L. Farkas - One of the best experts on this subject based on the ideXlab platform.
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RhoD 2 based measurements of intracellular calcium in the perfused mouse heart cellular and subcellular localization and response to positive inotropy
Journal of Biomedical Optics, 2001Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. Koretsky, Vazha Glonty, Joseph SuhanAbstract:We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye RhoD-2. In Langendorff perfused isolated mouse hearts, RhoD-2 is bolused through the perfusate, resulting in a 6.2+/-1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of RhoD-2 in the heart was done by taking the ratio of absorbance at 524 nm (RhoD-2 sensitive) to 589 nm (RhoD-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 microM). Electron microscopy was used to determine the subcellular localization of RhoD-2, by fixing RhoD-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain RhoD-2. These images demonstrated prominent cytosolic RhoD-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of RhoD-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 microM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686+/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199+/-215 and 544+/-53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.
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calcium measurements in perfused mouse heart quantitating fluorescence and absorbance of RhoD 2 by application of photon migration theory
Biophysical Journal, 2001Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye RhoD-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of RhoD-2 in the heart and measurement of the changes in quantum yield of RhoD-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with RhoD-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.
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Calibration of Ca2+ dissociation constant of RhoD-2 In vivo in perfused mouse heart using Mn2+ quenching
Biomedical Optical Spectroscopy and Diagnostics, 2000Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:We present an analytical model to characterize the calcium dissociation constant of the fluorescent indicator RhoD-2 in vivo in perfused mouse heart using manganese quenching fluorescence transients. We also provide the experimental results to validate this approach and determine that the calcium dissociation constant for RhoD-2 is 720nM.
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quantitative measurement of calcium transients in perfused mouse heart using RhoD 2 fluorometry
Advances in fluorescence sensing technology. Conference, 1999Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye RhoD-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37 degrees Celsius, and is stimulated at 8 Hz, with 100 (mu) g of RhoD-2 bolused through the perfusate. After a 25-minute washout period, approximately 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O2, thus minimizing interference due to changes in tissue oxygenation. Ca2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33 plus or minus 9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of RhoD-2 through the perfusate, the reflected absorbance between 524 nm (RhoD-2 sensitive) and 589 nm (RhoD- 2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATPase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368 plus or minus 68 nM and 654 plus or minus 164 nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
Guy A. Macgowan - One of the best experts on this subject based on the ideXlab platform.
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RhoD 2 based measurements of intracellular calcium in the perfused mouse heart cellular and subcellular localization and response to positive inotropy
Journal of Biomedical Optics, 2001Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. Koretsky, Vazha Glonty, Joseph SuhanAbstract:We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye RhoD-2. In Langendorff perfused isolated mouse hearts, RhoD-2 is bolused through the perfusate, resulting in a 6.2+/-1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of RhoD-2 in the heart was done by taking the ratio of absorbance at 524 nm (RhoD-2 sensitive) to 589 nm (RhoD-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 microM). Electron microscopy was used to determine the subcellular localization of RhoD-2, by fixing RhoD-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain RhoD-2. These images demonstrated prominent cytosolic RhoD-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of RhoD-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 microM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686+/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199+/-215 and 544+/-53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.
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calcium measurements in perfused mouse heart quantitating fluorescence and absorbance of RhoD 2 by application of photon migration theory
Biophysical Journal, 2001Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye RhoD-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of RhoD-2 in the heart and measurement of the changes in quantum yield of RhoD-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with RhoD-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.
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Calibration of Ca2+ dissociation constant of RhoD-2 In vivo in perfused mouse heart using Mn2+ quenching
Biomedical Optical Spectroscopy and Diagnostics, 2000Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:We present an analytical model to characterize the calcium dissociation constant of the fluorescent indicator RhoD-2 in vivo in perfused mouse heart using manganese quenching fluorescence transients. We also provide the experimental results to validate this approach and determine that the calcium dissociation constant for RhoD-2 is 720nM.
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quantitative measurement of calcium transients in perfused mouse heart using RhoD 2 fluorometry
Advances in fluorescence sensing technology. Conference, 1999Co-Authors: Guy A. Macgowan, Daniel L. Farkas, Alan P. KoretskyAbstract:We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye RhoD-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37 degrees Celsius, and is stimulated at 8 Hz, with 100 (mu) g of RhoD-2 bolused through the perfusate. After a 25-minute washout period, approximately 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O2, thus minimizing interference due to changes in tissue oxygenation. Ca2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33 plus or minus 9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of RhoD-2 through the perfusate, the reflected absorbance between 524 nm (RhoD-2 sensitive) and 589 nm (RhoD- 2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATPase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368 plus or minus 68 nM and 654 plus or minus 164 nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.