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Second harmonic imaging of chloroplasts using the two-photon laser scanning microscope
Authors:Ali Hussain Reshak  Vassilios Sarafis  Rainer Heintzmann
Institution:1. Institute of Physical Biology, University of South Bohemia, Nove Hrady 37333, Czech Republic;2. Institute of System Biology and Ecology, Academy of Sciences, Nove Hrady 37333, Czech Republic;3. School of Integrative Biology, University of Queensland, St. Lucia 4072, Queensland, Australia;4. School of Physics, University of Queensland, St. Lucia 4052, Queensland, Australia;5. Physics School, University of Melbourne, VIC 3010, Australia;6. University of Western Sydney, University of Western Sydney, South Penrith, NSW, Australia;7. King’s College London, Randall Division of Cell and Molecular Biophysics, New Hunts House, Guy’s Campus, London SE1 1UL, UK;1. Northwest Research Institute, 732 Caroline St., Port Angeles, WA, United States;2. Department of Mathematics, University of Ioannina, 451 10 Ioannina, Greece;1. Institute of Solid State Physics of RAS, Chernogolovka, Moscow region 142432, Russia;2. Physics Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;3. Chemical Engineering Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;1. Department of Conservative and Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University, Amsterdam, The Netherlands;2. Van ''t Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam, The Netherlands;1. Université de Lorraine, LIEC, Faculty of Sciences and Technology, BP 70239, 54506 Vandoeuvre-les-Nancy Cedex, France;2. CNRS, LIEC, Faculty of Sciences and Technology, BP 70239, 54506 Vandoeuvre-les-Nancy Cedex, France;3. Laboratoire de chimie analytique Bio-Inorganique et Environnement (LCABIE), Université de Pau et des Pays de l’Adour/CNRS, UMR 5254 IPREM, Hélioparc, 2 Avenue du Président Angot, 64053 Pau Cedex 9, France;4. Research and Development Division, Andra, Parc de la Croix Blanche, 1/7 rue Jean Monnet, 92298 Châtenay-Malabry Cedex, France;1. Frontier Research Center, Canon, Ohta-ku, Tokyo 146-8501, Japan;2. Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan
Abstract:BackgroundSecond harmonic information reveals information about the structure of spatially oriented structures with an asymmetry. We study the second harmonic images of the grana and intergranal parts of chloroplasts in the leaves of the shade moss Plagiomnium affine.ResultsA two-photon microscope with blocking filters to suppress fluorescence generated both forward and backward second harmonic illumination to be captured. In the starch free chloroplasts of the single cell layered laminae strong second harmonic generation (SHG) from the granal regions was emitted. Upon illumination the chloroplasts changed their orientation affecting the SHG signal. Chloroplast is reoriented upon illumination.ConclusionsSHG signals were successfully obtained from the grana without any possible confusion from SHG starch grains due to their absence.
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