Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS.
Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1nm was used for imaging the ultrastructure of algae cells which have diameters of 5-10μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast.
Université de Pau et des Pays de l'Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les, Matériaux (IPREM), UMR 5254, 64000 Pau, France.
Florent
F
Penen
Université de Pau et des Pays de l'Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les, Matériaux (IPREM), UMR 5254, 64000 Pau, France.
Julien
J
Malherbe
Université de Pau et des Pays de l'Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les, Matériaux (IPREM), UMR 5254, 64000 Pau, France.
Marie-Pierre
MP
Isaure
Martin-Luther-Universität Halle-Wittenberg, Institute for Biochemistry and Biotechnology, Plant Biochemistry, Weinbergweg 22, 06120 Halle (Saale), Germany.
Dirk
D
Dobritzsch
Martin-Luther-Universität Halle-Wittenberg, Institute of Biology, Plant Physiology, Weinbergweg 22, 06120 Halle (Saale), Germany.
Ivo
I
Bertalan
Université de Bordeaux, Bordeaux Imaging Center UMS 3420 CNRS - US4 INSERM, Pôle d'imagerie électronique, 146 rue Léo Saignat, 33076 Bordeaux, France.
Etienne
E
Gontier
Université de Pau et des Pays de l'Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les, Matériaux (IPREM), UMR 5254, 64000 Pau, France; Université de Bordeaux, UF Sciences de la Terre et Environnement, Allée G. Saint-Hillaire, 33615 Pessac, France.
Philippe
P
Le Coustumer
Université de Pau et des Pays de l'Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les, Matériaux (IPREM), UMR 5254, 64000 Pau, France. Electronic address: dirk.schaumloeffel@univ-pau.fr.
Dirk
D
Schaumlöffel
D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D018629:Spectrometry, Mass, Secondary Ion; D013052:Spectrometry, X-Ray Emission; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D014131:Trace Elements / Q000032:analysis* / Q000378:metabolism
D014131:Trace Elements
Chlamydomonas; Correlative imaging; Nanoscale; RF plasma ion source; Single cells; Trace metals