Biophysical techniques in photosynthesis

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  • Self-assembly of nanostructured polymetallaynes polymer. Ilaria Fratoddi, Christoph Gohlke, Cesare Cametti, Marco Diociaiuti, and Maria Vittoria Russo. Polymer. 2008; 49(15): 3211-3216.
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    Organometallic conjugated polymers containing transition metal centers in the main chain (polymetallynes) , with general formula -[M-(PBu3)2-C=C-X-C=C-]n- with M = Pt(II) and Pd(II), X= organic conjugated spacer, namely poly[1,1'-bis(ethynyl)-4,4'-biphenyl-(bis-tributylphosphine)Pd(II)] (Pd-DEBP), poly[1,1'-bis(ethynyl)-4,4'-biphenyl-(bis-tributylphosphine)Pt(II)] (Pt-DEBP) and poly-[1,4-bis(ethynyl)-2,5-dihexadecyloxy)benzene-bis(triphenylphosphine)platinum(II)] (Pt-BOB) were synthesized and fully characterized. The polymeric compounds were cast deposited onto glass substrates and their morphologies studied by means of SEM (Scanning Electron Microscopy) and EF-TEM (Energy Filtered-Transmission Electron Microscopy). The formation of nanostructured fibrils with diameters in the range 100-300 nm was revealed by SEM. EF-TEM images showed that the fibers are made of hollow nanotubes, randomly oriented, with external diameter of about 6-7 nm.

  • Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient. Oliver Holub, Manfredo J Seufferheld, Christoph Gohlke, Govindjee, Gregor J Heiss, and Robert M Clegg. J Microsc. 2007; 226(2): 90-120.

    Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25–35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a ‘dimmer switch’. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant.
    The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The above results are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

  • Fluorescence lifetime-resolved imaging: measuring lifetimes in an image. Robert M Clegg, Oliver Holub, and Christoph Gohlke. In Biophotonics, Part A (Methods in Enzymology, Vol. 360). By G Marriott and I Parker (Editors). Academic Press, pp. 509-42, 2003. ISBN 012182263X

    We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation. Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation--and therefore affect the measured fluorescence lifetime–that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging. FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging extends considerably the potential information that can be derived from a fluorescence experiment. Our purpose has been to arouse an appreciation for the broad application of fluorescence lifetime-resolved measurements in imaging. We have given only general design characteristics of the instrumentation and discussed the characteristics that distinguish imaging from the single channel lifetime-resolved measurements. We have not provided details of the instrumentation or the presented many examples. These are available in the literature, and given in the references, and they are continually and rapidly growing.

  • Fluorescence lifetime imaging (FLI) in real-time - a new technique in photosynthesis research. Oliver Holub, Manfredo J Seufferheld, Christoph Gohlke, Govindjee, and Robert M Clegg. Photosynthetica. 2000; 8(4): 581-599.

    We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chi a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves.

  • Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes. Gyorgy Vámosi, Christoph Gohlke, and Robert M Clegg. Biophys J. 1996; 71(2): 972-94

    Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.

  • Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer. Christoph Gohlke, Alastair I H Murchie, David M J Lilley, and Robert M Clegg. Proc Natl Acad Sci USA. 1994; 91(24): 11660-4

    Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

  • A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements. Thomas Tuschl, Christoph Gohlke, Thomas M Jovin, Erick Westhof, and Fritz Eckstein. Science. 1994; 266(5186): 785-9.
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    For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozy

  • Diplomarbeit. Christoph Gohlke. Georg-August-Universität Göttingen, 1993.
  • . Gyorgy Vámosi, Christoph Gohlke, Alastair I H Murchie, David M J Lilley, and Robert M Clegg. 11th International Biophysics Congress, Budapest, Hungary, July 25-30, 1993.

    Fluorescence studies, especially fluorescence resonance energy transfer (FRET), have aided in determining conformations of complex molecular structures of nucleic acids. Oligonucleotides are synthesized, covalently labeled with dyes, and assembled to form specific structures. For instance, we have applied sensitive fluorescence methods to probe the helical structure of simple specifically labeled DNA duplexes and to investigate more complex structures, such as four-way DNA junctions (a model in solution, of the Holliday genetic recombination junction) and bulged DNA molecules (duplex structures with extra nucleotides in one of the strands). FRET, fluorescence anisotropy and intensity determinations, and kinetic measurements of helix-coil transitions provide us with molecular scale information regarding the conformations of these structures, and the conformational changes ensuing upon perturbations of the molecular environment. Examples will be given and the techniques will be discussed.

  • Versatile rapid lifetime imaging. Glen I Redford, Christoph Gohlke, and Robert M Clegg. 47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003. Biophys J. 2003; 84 (2 Suppl. S): 584, 2860-Pos/B459.

    A full-field fluorescence lifetime instrument using a homodyne frequency domain method will be presented. This instrument captures and displays real-time lifetime images at rates higher than 30 frames per second. It can be adapted to a microscope or an endoscope. Multiple frequencies are easily implemented. Immediate feedback and ease of use have been emphasized so that the device to can also be used for clinical diagnostic purposes. The instrument has been designed so that the benefits of lifetime imaging can be employed conveniently wherever standard fluorescence imaging is used. Data is shown for biological and biotechnological samples.

  • Imaging in skin and plants: using photons and fluorescence lifetimes to find the molecules and quantify the information. Kerry Hanson, Oliver Holub, Christoph Gohlke, Nicholas P Barry, Martin J Behne, Enrico Gratton, and Robert M Clegg. The 8th Biophysical Society Annual Meeting and Frontier Biophysics Symposium, Taiwan, ROC, 2002.

    Due to sophisticated technological advances and innovative applications of new technology, numerous optical imaging techniques are being developed that are highly informative, quantitative, flexible and which can be applied directly to biological and biophysical studies and to medical diagnostics. Fluorescence imaging is one of the fastest growing areas. Many measurements that were previously limited to cuvette type studies are now being incorporated into fluorescence microscopes and into medical diagnostics (such as the endoscope). This lecture will describe two techniques 1) “Fluorescence Lifetime-resolved Imaging” (FLI - where every pixel of the image contains the lifetime-resolved information) and its application to several biological areas, and 2) two-photon scanning imaging applied to skin diagnostic measurements. A brief general description of the various methods in our laboratory will be presented. The reason for making fluorescence lifetime measurements in images will be discussed. The necessity for real-time imaging is stressed, and the performance characteristics of our latest fast-FLI instrument will be briefly explained (including methods for rapid, informative display of complex data). Several biological problems (tumor diagnostics in cellular/biomedical biology, lifetime imaging measurements of chlorophyll a fluorescence in photosynthesis, and tissue imaging) that can be profitably studied by fast-FLI will be introduced. The unique information available will be emphasized. We will also discuss measurements of two-photon excitation imaging in tissue. Recent results of quantitative determinations of reactive oxygen species (caused by sun exposure) in biological tissue will be presented.

  • Fast macroscopic chlorophyll fluorescence lifetime imaging of apple fruit skin. Martin vandeVen, Oliver Holub, Christoph Gohlke, Govindjee, Roland Valcke, Marcel Ameloot, and Robert M Clegg. 46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002. Biophys J. 2002; 82(1): 502a.

    Early detection of fruit skin storage diseases like bitterpit and storage scald is aided by macroscopic imaging of healthy and diseased tissue. Fast, whole area detection is necessary for quickly assessing fruit quality and for predicting storage potential. Macroscopic fluorescence lifetime imaging enhances contrast w.r.t. steady-state images and reduces geometrical effects. By monitoring photosystem II Chlorophyll a fluorescence it provides quick physiological information over an area not practically accessible by confocal (TPE) microscopy (Biophys. J., 80, 159a). Here we report using a fast-acquisition homodyning phase and modulation imaging fluorimeter (Biophys. J. 80, 169a, 428a) on a 1.8 cm diameter fruit skin area. Red and green/yellow sides of Malus Domesticus Borkh. x Golden Delicious, Jonagold and Granny Smith with and without surface defects were examined with a 690DF40 emission filter. A 6 μm single-mode fiber-optic directed the AOM modulated 488 nm laser light. Operating frequency was 80.652 MHz and total power delivered was 0.28 mW. Time progression of total intensity, phase and modulation images of diseased and healthy tissue are presented. Differences in observed Chlorophyll fluorescence lifetimes range from 0.4 and 1.8 nsec. Kautsky-effect fluorescence induction effects on imaging are discussed. This work was supported by the “Fonds Slimme Regio” of the Province of Limburg, BOF, and NIH, PHS 5 P41 RRO3155.

  • FliFast: Software for fast fluorescence lifetime-resolved image acquisition with concurrent analysis and visual feedback. Christoph Gohlke, Oliver Holub, and Robert M Clegg. 45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001. Biophys J. 2001; 80(1 Pt 2): 169a, 656.54-Pos.

    Fluorescence lifetime-resolved imaging FLI provides indispensable, valuable information than is not available from steady-state fluorescence image measurements. In general, because of the complexity of the analysis compared to the display of simple intensity images, FLI measurements have required much longer times to process and display. However, many biological and medical applications using fluorescence imaging require real time acquisition, processing and display. Real-time operation is necessary for following dynamic biological events, or carrying out medical diagnostics. We have developed a real-time FLI system for a variety of imaging applications. The instrument uses rapid frequency-domain data acquisition hardware; however, here we demonstrate software that specifically enables the real-time processing and highly informative, convenient and easy-to-understand display of the lifetime-resolved fluorescence information. A menu of possibilities is provided to the operator to assist in the on-line interpretation and control of real-time experiments and rapid events. The programs have been streamlined in order to make the multiple-parameter, lifetime-resolved information available to the user, and provide a continual up-to-date view of the statistics. NIH, PHS 5 P41 RRO3155

  • Application of real-time fluorescence lifetime-resolved imaging in photosynthesis: studies of maize leaves (Zea Mays), small mustard leaves (Arabidopsis Thaliana), and of individual wild-type and mutant cells of the green alga Chlamydomonas Reinhardtii. Oliver Holub, Manfredo Seufferheld, Christoph Gohlke, Govindjee, and Robert M Clegg. 45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, February 17-21, 2001. Biophys J. 2001; 80(1 Pt 2): 428a, 1819-Pos.

    Chlorophyll (Chl) a fluorescence from leaves of Zea Mays, Arabidopsis Thaliana and from single cells of wild type (WT) and non-photochemical quenching mutants (NPQ1 and NPQ2) of C. Reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved microscope images. Objects are imaged using the frequency domain phase and modulation technique in homodyne mode. The laser light is modulated at a high frequency. The fluorescence image is focused onto a modulated image intensifier and the phase-resolved images are captured on a fast CCD camera, sequentially transferred to a PC computer and displayed in real-time. Rapid measurements are necessary during the fluorescence transient maximum (the P-level) following initial illumination. The observed lifetime heterogeneity is in accordance with photosynthetic functionality. The NPQ1 and NPQ2 mutants (Niyogi et al., 1997) used in this work accumulate violaxanthin and zeaxanthin, respectively. A decrease in the lifetime of Chl a in NPQ2 was observed consistent with quenching due to Förster energy transfer from Chl a to zeaxanthin. Time-resolved fluorescence microscopy of the NPQ mutants is expected to provide new information on the role of the Xanthophyll cycle in the non-photochemical quenching process in single algal cells. This work was supported by NSF, DBI 96-0240 and NIH, PHS 5 P41 RRO3155

  • Real-time fluorescence lifetime-resolved images of individual cells of wild type and NPQ mutants of Chlamydomonas reinhardtii. Oliver Holub, Manfredo Seufferheld, Christoph Gohlke, Robert M Clegg, and Govindjee. 9th International Conference on the Cell and Molecular Biology of Chlamydamonas, Amsterdam, The Netherlands, 2000.

    The chlorophyll a (Chl a) fluorescence from single cells of wild type (WT) and non-photochemical quenching (NPQ) mutants (NPQ1 and NPQ2) of Chlamydomonas reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved images (see Instrumentation). The mutants NPQ1 and NPQ2 used in this work (Niyogi et al., 1997) , accumulate violaxanthin and zeaxanthin, respectively. Fluorescence transient studies (see poster by Seufferheld et al., 2000) show strong quenching of Chl a fluorescence in the NPQ2 mutant in comparison to the WT and the NPQ1 mutant. If the quenching is due to Förster transfer from Chl a to zeaxanthin, then the lifetime of Chl a in the NPQ2 mutant will decrease. The time-resolved fluorescence microscopy of the NPQ mutants can therefore be used to study the role of the Xanthophyll cycle in the non-photochemical quenching process in single cells. This work is supported by NIH PHS 5 P41-RRO3155 and NSF DBI96-0240.

  • Want to know something about your fluorescent samples that optics cannot resolve? Long working-distance stage-scanning instrument for Real-Time Fluorescence Lifetime-Resolved Imaging. Oliver Holub, Christoph Gohlke, Manfredo Seufferheld, Govindjee, and Robert M Clegg. 44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000. Biophys J. 2000; 78(1 Pt 2), 1464-Pos.

    We present a new high-speed instrument for rapidly measuring fluorescence lifetime-resolved images of large as well as small objects in real time (up to 55 phase-resolved images per second). This instrument employs stage-scanning while measuring lifetime-resolved fluorescence images in the phase and modulation, homodyne mode. Long working-distance objectives (5 to 100 times magnification) are used for illuminating the sample with HF-modulated laser light; the sample is placed on a piezo-controlled xy-scanning stage; all pixels of the fluorescence image are imaged simultaneously and projected onto a HF-modulated image intensifier. A fast CCD camera captures the incrementally phase-delayed modulated images, that are acquired, processed and displayed in real-time on a PC computer. Light modulation (of the CW laser) is achieved with a standing wave acousto optical modulator. We demonstrate the functionality and use of the instrument with different examples. The instrument is especially useful for rapidly scanning even larger extended objects while simultaneously displaying lifetime-resolved images as rapid as (or faster than) video mode. Such rapid data acquisition is also essential for measuring lifetime-resolved images that are rapidly changing their fluorescence characteristics due to e.g. chemical reactions, movement or photobleaching. This work is supported by NIH PHS 5 P41-RRO3155 and NSF DBI96-0240

  • Photophysics of 5-carboxytetramethylrhodamine linked to the 5'-end of ss & ds DNA molecules. Gyorgy Vámosi, Christoph Gohlke, and Robert M Clegg. XIIth International Biophysics Congress, Amsterdam, The Netherlands. Prog Biophys Mol Biol. 1996; 65(Suppl 1): 76, P-B1-31.

    Purpose: To determine the photophysical properties of tetramethylrhodamine (TMRh) conjugated to single-stranded (ss) and doublestranded (ds) DNA oligomers.
    Methods: TMRh was coupled (via a C6 linker) to the 5' end of oligonucleotides. Fluorescence measurements on ds and ss TMRh-labeled DNA species were made over a large range of NaCl concentrations and temperatures, and in the presence of added ethanol. Static and lifetime-resolved fluorescence results are presented.
    Results: Three well-defined states of TMRh-DNA exist at lower (cl00 mM) and at higher ionic strengths. Two states fluoresce with lifetimes of 0.5 - 1.5 ns and 2.5 - 4.5 ns; a third state does not fluoresce (dark species). All three states of TMRh are present on ds and ss DNA. It is possible to extract thermodynamic parameters related to the interaction of the TMRb with the nucleic acid molecule, and of ions with the TMRh-DNA complex.
    Conclusions: Three states of TMRh conjugated to DNA are present. The distribution of TMRh among these states depends on the temperature, ionic strength and polarity of the solvent, and on the ss/ds state of the oligomers

  • A 3-dimensional model for the hammerhead ribozyme from fluorescence resonance energy-transfer measurements. Thomas Tuschl, Christoph Gohlke, Thomas M Jovin, Eric Westhof, and Fritz Eckstein. Keystone Symposia on Molecular & Cellular Biology, Santa Fe, New Mexico, 1995. J Cell Biochem. 1995; 59(S19A): 218.

    For the understanding of the catalytic function of the hammerhead ribozyme, a three-dimensional model is essential. Synthetic constructs were labeled with fluorescence donor fluorescein and acceptor tetramethylrhodamine located at the ends of one of the two strands constituting the ribozyme molecule. Distance relationships were established between different pairs of helices of various length. The FRET efficiencies were determined and correlated with measurements of a reference set of labeled RNA duplexes. Based on vector algebra analysis, FRET efficiencies were predicted as a function of the relative helical orientations in the various ribozyme constructs and compared with the experimental values. The data were most consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. Using these orientational constraints and information from biochemical studies, molecular modeling was carried out and led to a three-dimensional structure of the complete ribozyme.

  • Structure of the hammerhead ribozyme from fluorescence resonance energy-transfer measurements. Thomas Tuschl, Christoph Gohlke, Thomas M Jovin, Eric Westhof, and Fritz Eckstein. FASEB J. 1994; 8(7): A1324-A1324.

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