Use of fib‐sem to address the ultrastructure of the periplastidial and internal chloroplast compartments of the diatom Phaeodactylum tricornutum
Authors: Flori Serena , Jouneau Pierre‐Henri , Gallet Benoit , Moriscot Christine , Estrozi Leandro , Petroutsos Dimitris , Breyton Cécile , Schoehn Guy , Maréchal Eric , Finazzi Giovanni , Falconet Denis 
Published: December 2016

Use of FIB‐SEM to address the ultrastructure of the periplastidial and internal chloroplast compartments of the diatom
Phaeodactylum tricornutum

Serena Flori
1
, Pierre‐Henri Jouneau
2
, Benoit Gallet
3
, Christine Moriscot
3
, Leandro Estrozi
3
, Dimitris Petroutsos
1
, Cécile Breyton
3
, Guy Schoehn
3
, Eric Maréchal
1
, Giovanni Finazzi
1
and Denis Falconet
1

1
Laboratoire de Physiologie Cellulaire et Végétale; UMR5168 CNRS / Univ. Grenoble Alpes / CEA / UMR1417 INRA; Institut de Biosciences et Biotechnologies de Grenoble (BIG), France.

2
Laboratoire d'Etudes des Matériaux par Microscopie Avancée (LEMMA); Institut Nanosciences et Cryogénie; Service de Physique des matériaux et Microstructures; CEA‐Grenoble, France.

3
UMR5075, Institut de Biologie Structurale (IBS), Grenoble, France.

Focused Ion Beam Scanning Electron Microscope (FIB‐SEM) is a state‐of‐the‐art advanced scanning electron microscope integrated with high‐resolution focused ion beam milling that enables photographic, chemical, and structural analysis of many inorganic and organic samples. Among different features FIB‐SEM allows 3D tomography with nanometer‐scale resolution thus becoming an instrument of choice for obtaining 3D information at the EM level.

In this study, FIB tomography has been realized in a Zeiss NVision 40 dual‐beam microscope to reveal the ultrastructure of the unique chloroplast present in the diatom
Phaeodactylum tricornutum,
a member of these key ecological players in oceans for CO2 sequestration via photosynthesis. A striking feature of diatoms is their sophisticated ultrastructure, inside highly packed cells, including a chloroplast bounded by four membranes known as 'secondary' plastid when 'primary' plastids as those present in plant cells have two membranes. Although their photosynthetic apparatus is similar to that of plants, photosynthesis takes place in a different plastid environment, where membranes are note differentiated in appressed (grana stacks) and non‐appressed regions (stroma lamellae).

Analyze of 4 nm ultrathin sections (figure 1) of disrupted
Phaeodactylum tricornutum
allows the detection of membrane connectivity in the three dimensions and thus the ultrastructure scanning of single organelle (chloroplast, mitochondrion or nucleus). The inner (iEM) and outer (oEM) envelope membranes of the symbiont's chloroplast together with the two outermost membranes: the chloroplastic endoplasmic reticulum membrane (cERM), which is continuous with the outer nuclear envelope and the periplastidial membrane (PPM) are well defined. Images reveal the presence of a vesicular network (VN) between the oEM and the PPM delineating the remaining of the symbiont cytoplasm and referred as the periplastidial compartment (PPC) (figure 2) [1].

Reconstitution of the three‐dimensional architecture of the chloroplast in
Phaeodactylum tricornutum
using focused ion beam scanning electron microscopy allows proposing a structural model for the arrangement of photosynthetic complexes in this chloroplast. This model challenges the classic view of the organization of the photosynthetic membranes in chloroplasts derived form a secondary endosymbiosis, and accounts for partitioning of absorbed light between the photosystems, without restraining electron flow capacity, as required for optimum photosynthesis (figure3) [2].