Highly Sensitive Pulsed Digital Holography for Built-in Defect Analysis with a Laser Excitation.

A highly sensitive method is presented for noninvasive defect analysis on thin structures with a Q-switched double-pulsed ruby laser with frequency doubling (347 nm). In our research we feature an all-optical arrangement, where a focused laser pulse derived from the same ruby laser (694 nm) acts as a built-in synchronous excitation source for digital holographic interferometry. The recordings are made with a CCD camera for capturing two holograms (two states of the specimen) corresponding to the two UV laser pulses with a short time separation (10-50 mus). Subtraction of the phase distribution in two digital holograms gives a fringe phase map that shows the change in deformation of the specimen between the recordings. The advantage of the proposed method is two fold. First, the use of a shorter wavelength results in a higher sensitivity. Second, owing to the induced synchronous built-in optical excitation, the specimen is not subjected to any external physical excitation devices. Experimental results are presented on identification and evaluation of defects in thin metal sheets.

Comparative study of various endoscopes for pulsed digital holographic interferometry.

A comparison of several endoscopes as object image carriers in pulsed digital holography is presented. Three multicore flexible fiber endoscopes of different spatial resolution and one rigid endoscope are investigated. The four endoscopes are integrated in a setup for the recording of digital holograms on a CCD camera. A double-pulsed ruby laser is used as the light source. A spatial carrier is introduced by an off-axis reference beam, which permits quantitative evaluation of the phase difference between two holograms recorded with a short time separation (5-600 micros). From reported studies it may be inferred that the quality of the phase maps so derived from digital holographic interferometry has a strong correlation to the spatial resolution of the multicore fiber used in these endoscopes. With the endoscopic technique combined with pulsed digital holography a number of useful applications (in areas such as medical endoscopy, micromechanics, and microelectronics) are envisaged for which access to the objects of interest is otherwise difficult.

Pulsed digital holographic interferometry for dynamic measurement of rotating objects with an optical derotator.

A method for measuring dynamic deformations of rotating objects with pulsed digital holography is described. An optical derotator is used to compensate for the rotation. A CCD camera is used to record two holograms with a short time separation (20 mus). Results of deformations between the recordings are obtained after subtraction of the phase distribution between the two digital holograms. Fringe phase maps of the phase subtraction of two holograms compensated by the derotator and recorded with a Q-switched double-pulsed ruby laser are presented. A flat disk and the blades of a fan were investigated. We used an optical arrangement that allowed us to improve laser illumination and energy efficiency. Experimental results on quantitative evaluation of dynamical out-of-plane deformations are presented.

Influence of peroxisome proliferator-activated receptor alpha on ubiquinone biosynthesis.

The control of ubiquinone biosynthesis by peroxisome proliferators was investigated using peroxisome proliferator activated receptor alpha (PPARalpha)-null mice. Administration of 2-(diethylhexyl)phthalate to control mice resulted in elevated ubiquinone levels in the liver, while dolichol, dolichyl-P and cholesterol concentrations remained unchanged. In PPARalpha-null mice, the level of these lipids were similar to control levels and administration of the peroxisome proliferator did not increase the levels of ubiquinone. The increase in ubiquinone levels was the result of increased synthesis. Induction was most pronounced in liver, kidney and heart, which have relatively high levels of PPARalpha. When the tissue concentration of hydrogen peroxide was elevated by inhibition of catalase activity with aminotriazole, the amount of ubiquinone was not increased, suggesting that the induction of ubiquinone synthesis occured through a direct mechanism. The activities of branch-point enzymes FPP-synthase, squalene synthase, cis-prenyltransferase, trans-prenyltransferase and NPHB-transferase were substantially increased in control but not in PPARalpha-null mice after treatment with peroxisome proliferators. These data suggest that the induction of ubiquinone biosynthesis after administration of peroxisome proliferators is dependent on the PPARalpha through regulation of some of the mevalonate pathway enzymes.

Pulsed digital holography for deformation measurements on biological tissues.

Digital holograms are recorded of biological tissues by use of a Q-switched double-pulsed ruby laser. An image-plane digital holography setup is used with a CCD camera for capturing two holograms with a short time separation (20-800 micros). Subtraction of the phase distribution in two digital holograms yield a fringe phase map that shows the change in deformation of the tissue surface between the recordings. Experiments are performed on tissue from a pig that was excited by a short-shock pulse and on a human hand that was excited by sinusoidal stimulation. Results when the object is imaged through an endoscope are also presented. The technique could be an approach for measuring parameters like elasticity on biological tissues.

Simultaneous three-dimensional dynamic deformation measurements with pulsed digital holography.

The three deformation components x, y, z of a vibrating object are measured simultaneously by use of digital holography with a double-pulse ruby laser source. The object is illuminated from three different directions, each optically path matched with three reference beams such that three independent digital holograms are formed and added incoherently in one single CCD image. The optical phase difference between the two recordings taken for each hologram is quantitatively evaluated by the Fourier-transform method so that a set of three phase maps is obtained, representing the deformation along three sensitivity vectors. The total object deformation is obtained as a vector resultant from the data of the three phase maps. To give the full three-dimensional (3-D) description, the shape of the object is measured by the two-wavelength contouring method. Experiments are performed with a cylinder as the test object, transiently and harmonically excited. The 3-D deformation and shape measurement results are presented graphically.

Reduced cholesterol accumulation and improved deficient peroxisomal functions in a murine model of Niemann-Pick type C disease upon treatment with peroxisomal proliferators.

Niemann-Pick type C disease is an inherited disorder characterized by lysosomal accumulation of cholesterol and the mutant gene has recently been identified. The predicted gene product is a transmembrane protein showing homology to proteins involved in the regulation of cholesterol homeostasis, such as 3-hydroxy-3-methylglutaryl-coenzyme A and the sterol regulatory element binding protein cleavage-activating protein. Recent investigations have established a peroxisomal deficiency, which raised the question of whether peroxisomal proliferation could affect this cholesterol-processing error. Mutant mice with Niemann-Pick type C disease were treated with the peroxisomal inducer perfluorooctanoic acid, which increased peroxisomal beta-oxidation and catalase activity to the same level as in control mice. Not only the peroxisomal, but also the lysosomal malfunctions were corrected and the cholesterol content was decreased. Clofibrate, another peroxisomal inducer, restored both peroxisomal enzyme activities and ubiquinone content. It appears that in Niemann-Pick type C disease treatment with appropriate peroxisomal inducers restores basic cellular functions, indicating a relationship between peroxisomes and cholesterol homeostasis, and thereby may effectively interfere with the development of the disease.

Alterations in the biosynthesis of cholesterol, dolichol and dolichyl-P in the genetic cholesterol homeostasis disorder, Niemann-Pick type C disease.

The biosynthesis of cholesterol, dolichol and dolichyl-P were investigated in a murine model of Niemann-Pick type C disease using both in vitro and in vivo systems. In vivo incorporation of [3H]mevalonate into squalene, dolichol and dolichyl-P decreased. The amount of dolichyl-P was elevated due to a decrease in the rate of degradation. Labeling of squalene and cholesterol of liver homogenates in vitro was decreased in the diseased mice and a lowering of microsomal activities of both HMG-CoA reductase and squalene synthase were also observed. In experiments with brain homogenate, decreased [3H]mevalonate labeling of squalene, cholesterol and dolichol was found in vitro. The decreases in cis-prenyltransferase and squalene synthase activities were observed at a very early phase of the disease. In contrast to the decreased biosynthesis of cholesterol observed in vitro, the labeling of total liver cholesterol was found to be increased in Niemann-Pick type C liver upon in vivo investigation, possibly due to the accumulation of this lipid as a result of a deficient transport process. In the brain, where in vivo labeling reflects only biosynthesis, a decreased rate of cholesterol synthesis was demonstrated.

Tomographic reconstruction of transient acoustic fields recorded by pulsed TV holography.

Pulsed TV holography combined with computerized tomography (CT) are used to evaluate the three-dimensional distribution of transient acoustic fields in air. Experiments are performed with an electrical discharge between two electrodes as the sound source. Holograms from several directions of the acoustic field are recorded directly onto a CCD detector by use of a double-pulsed ruby laser as the light source. Phase maps, representing projections of the acoustic field, are evaluated quantitatively from the recorded holograms. The projections are used for the CT reconstruction to evaluate the pressure-field distribution in any cross section of the measured volume of air.

Phase evaluation and speckle averaging in pulsed television holography.

Double-pulsed (image-plane) TV holograms of transient bending waves in plates are recorded on separate frames in a CCD camera. A small angular offset between the reference and object beams, giving a spatial-frequency bias to the recorded pattern, permits quantitative evaluation of the phase of the interference. The Fourier spectrum of the image exhibits distinct parts that can be filtered out and inverse transformed to yield the phase information. Three different apertures of the imaging system are tested: a single slit, a double slit, and a three-hole aperture. Spatial speckle averaging is possible in the cases of the double-slit and three-hole apertures.

Peroxisomal impairment in Niemann-Pick type C disease.

Niemann-Pick type C disease (NPC) belongs to the group of lysosomal storage diseases characterized by an accumulation of cholesterol and sphingomyelin. Using a mutant mouse strain, enzymatic markers for lysosomes, mitochondria, microsomes, and peroxisomes were investigated in the liver and brain. Aside from lysosomal changes, we found a sizable decrease of peroxisomal beta-oxidation of fatty acids and catalase activity in the brain and liver. Isolated peroxisomes displayed a significant decrease of these enzyme activities. Furthermore, the only phospholipid change in brain was a decreased content of the plasmalogen form of phosphatidylethanolamine, and the dimethylacetal pattern was also modified. The electron microscopical appearance of peroxisomes did not display any large changes. The defect of peroxisomal enzymes was already present 18 days before the onset of the disease. In contrast, the lysosomal marker enzyme increased in activity only 6 days after appearance of the symptoms. The events of the studied process have previously been considered to be elicited by a lysosomal deficiency, but this study demonstrates disturbances similar to those in a number of peroxisomal diseases. It appears that the peroxisomal impairment is an early event in the process and could be a factor in the development of Niemann-Pick type C disease.

Changes in the levels of dolichol and dolichyl phosphate in a murine model of Niemann-Pick's type C disease.

The distributions of mevalonate pathway lipids in various organs of a mouse strain used as a model for Niemann-Pick's type C disease were analyzed. Extensive accumulation of cholesterol was observed in all tissues with the exception of the brain, where the content of this lipid was decreased. The changes in total dolichol contents of most organs varied from a 50% decrease in the lung to a twofold increase in kidney and heart. There was relative enrichment of longer-chain dolichols, but no increase in the relative amount of alpha-unsaturated polyprenols was observed. The levels of dolichyl phosphate in all organs were increased, and most of this lipid was associated with bound oligosaccharides or proteins. Ubiquinone levels were largely unchanged. Subfractionation studies revealed that heavy and light lysosomes exhibited a 10-fold increase in cholesterol level, the amount of dolichol was decreased in lysosomes and increased in microsomes, and there was an increase in the dolichyl phosphate levels of all three of these subfractions. These results indicate that in diseased mice cholesterol accumulation in various organs is paralleled by an increase in the dolichyl phosphate concentration, whereas dolichol transport from the endoplasmic reticulum to lysosomes is inhibited.

Enzymes of the mevalonate pathway in rat liver nodules induced by 2-acetylaminofluorene treatment.

Certain enzymes of the mevalonate pathway have been investigated in persistent liver nodules induced in the rat by 2-acetylaminofluorene. In these nodules the dolichol level was increased 5-fold, the ubiquinone-9 content elevated 6-fold and the amount of cholesterol unchanged. Microsomal beta-hydroxy-beta-methylglutaryl-coenzyme A reductase activity was greatly increased compared to control liver tissue, which was also the case for the cytosolic farnesyl pyrophosphate synthase. A significant elevation of all-transgeranylgeranyl pyrophosphate synthase activity in the cytosol was also observed. The branch-point enzyme of microsomal dolichol synthesis, i.e. cis-prenyltransferase, was decreased in the nodules; whereas the activity of squalene synthase, the terminal regulating enzyme of cholesterol synthesis, remained unchanged. The dolichol species in nodular tissue were redistributed towards the longer chain length species. One factor regulating the chain length of the polyisoprene products formed in vitro was shown to be the ratio of the concentrations of isopentenyl pyrophosphate:farnesyl pyrophosphate employed. Other regulatory factors in the terminal steps of this biosynthetic pathway appear to determine the amounts and nature of the final isoprenoid compounds formed in vivo. In contrast to the microsomal trans-prenyltransferase activity, which was unchanged, the activity of nonaprenyl-4-hydroxybenzoate transferase, an enzyme participating in ubiquinone synthesis, was greatly elevated. The alterations observed in the activities of enzymes in the mevalonate pathway can at least partially explain the increased levels of dolichol and ubiquinone and the unchanged level of cholesterol found in liver nodules. It is reasonable to propose that this modified mevalonate metabolism will render nodular cells resistant to certain toxic factors and prone to cell proliferation.

Modulations in hepatic branch-point enzymes involved in isoprenoid biosynthesis upon dietary and drug treatments of rats.

Three branch-point enzymes of the mevalonate pathway, farnesyl pyrophosphate synthase, cis-prenyltransferase and squalene synthase were characterized in rat hepatic cytosol, microsomes and peroxisomes isolated from rats after treatment with peroxisome proliferators, inducers of the endoplasmic reticulum or modulators of lipid metabolism. Cholestyramine and phenobarbital induced primarily the cytosolic farnesyl pyrophosphate synthase, whereas clofibrate and phthalates elevated the corresponding peroxisomal activity. cis-Prenyltransferase activities in microsomes were induced 4-5-fold after clofibrate, phthalate and phenobarbital administration, but these same treatments affected the peroxisomal activity to only a limited extent. Squalene synthase activity in microsomes was completely abolished, but the peroxisomal activity was unaffected after administration of cholesterol. On the other hand, clofibrate and phthalate induced only the microsomal activities. Mevinolin treatment greatly increased peroxisomal and cytosolic farnesyl pyrophosphate synthase activities, but not the mitochondrial activity, and the cis-prenyltransferase activities were elevated in peroxisomes, but not in microsomes. These results demonstrate that the branch-point enzymes in cholesterol and dolichol biosynthesis at various cellular locations are regulated differentially and that the capacities of peroxisomes and the endoplasmic reticulum to participate in the synthesis of polyisoprenoid lipids is affected profoundly by treatment with different xenobiotics.

Effects of inhibitors of hydroxymethylglutaryl coenzyme A reductase on coenzyme Q and dolichol biosynthesis.

Inhibitors of hydroxymethylglutaryl coenzyme A reductase are used clinically to decrease blood levels of low-density lipoprotein cholesterol in hypercholesterolemic patients. However, little is known about the possible effects of these inhibitors on dolichol and cholesterol synthesis. Oral administration of mevinolin to rats was found here to decrease dolichol, dolichyl-P and coenzyme Q levels in the heart and skeletal muscle and to increase the hepatic dolichol level while decreasing the coenzyme Q content in this same organ. The amounts of dolichyl-P decreased in heart and muscle and increased in brain. Intraperitoneal administration also affected the levels of these lipids. The concentrations of blood lipids were not modified in the same manner as tissue lipids. Analysis of individual enzyme activities and of incorporation of [3H]acetate into various lipids of liver and brain slices demonstrated that both up- and down-regulation of different proteins occur in various tissues, resulting in modifications in lipid synthesis. Hypercholesterolemic patients were found to have high blood coenzyme Q levels, which are decreased upon pravastatin treatment, although they are still above control values. It appears that these HMG-coenzyme A reductase inhibitors do not selectively lower cholesterol levels, but that they also modify the dolichol and coenzyme Q content and synthesis both in the liver and various other tissues.

Half-life of ubiquinone-9 in rat tissues.

The half-life of ubiquinone-9 in various rat tissues was determined. Rats were injected intraperitoneally with [3H]mevalonate and the decay of radioactivity incorporated into ubiquinone-9 was followed using reverse-phase HPLC. The half-life varied between 49 h (testis) and 125 h (kidney).