focus on Microscopy & Microtechniques


Electron Microscopy Reveals Secrets of Nanocrystal-Assembly in Coral Skeletons


Renée van de Locht Department of Physics, The University of York, Heslington, York YO10 5DD, UK.


One of the most important examples of mineral forming organisms are corals, in particular reef-building corals, which play central roles in the marine ecology by hosting 25% of marine livestock as well as the economy via coastal protection, tourism and fi shery. The mechanical stability of coral colonies is caused by a massive accretion of a calcium carbonate based exoskeleton. We are interested in the relation between growth conditions, such as the chemical composition of seawater, the pH value and the temperature on the microstructure of the mineral deposited by the corals. For this purpose we focused on the details of the microstructure down to the atomic level to understand the patterns of assembly of the nanocrystalline building blocks constituting the skeleton. These studies were undertaken using state- of-the-art electron microscopy available at the University of York and form parts of a collaboration with world-leading research groups in the area of Marine Science at the University of Western Australia and the University of St Andrews.


Transmission Electron Microscopy (TEM)


Instrumentation TEM samples were predominantly analysed at the York Jeol Nanocentre utilising a JEM 2011 (LaB6 heated fi lament with an EDX detector for elemental analysis) and JEM 2200 FS (fi eld emission, double Cs aberration corrected with EDX detector for elemental analysis and an in-column omega fi lter for EELS and EFTEM) TEM operated at 200kV.


Additionally TEM analysis was performed at the Centre for Microscopy, Characterisation and Analysis (CMCA) at the University of Western Australia using a JEM 2100 TEM (LaB6 heated fi lament with an 11M pixel Gatan Orius digital camera and a Gatan Tridiem energy fi lter for EELS) operated at 120 and 200 kV and a JEM 3000 F FEGTEM (fi eld emission with a 1M pixel Gatan 694 MSC digital camera and a Gatan GIF2000 energy fi lter for EELS and EFTEM) operated at 300 kV.


Microscope conditions and beam damage; Electron microscopy


During electron microscopy image or data acquisition, beam damage and sample contamination should be considered. Damage caused by elastic interactions can affect the original structure and/or chemistry of the specimen by a variety of phenomena such as the breaking of chemical bonds, the displacement of atoms, creating point defects or simply the heating caused by phonons interacting with the sample [1]. Contamination is caused by the deposition of carbonaceous material due to hydrocarbons that are present in the TEM chamber reacting with electrons from the beam forming hydrocarbon ions and condensing on the irradiated specimen surface [2]. In case of biogenic samples a hydrocarbon organic fi lm is often naturally present on the specimen itself, which greatly enhances this phenomenon. Especially during analytical EM: EDX and EELS, beam damage is problematic as the beam is often condensed on a spot for several tens of seconds’ acquisition time. This can cause severe structural damage: removal of material, and re-deposited together with the previously mentioned hydrocarbons on the specimen surface. Beam damage also occurs during high resolution imaging as the high magnifi cation (>400 000 x) acts as a condensed beam on a small surface area, this often results in damage to the crystal lattice fringes (Figure 1).


To reduce beam damage imaging and diffraction is performed using predominantly a LaB6 fi lament TEM instead of a fi eld emission gun, which has a higher intensity. Furthermore small condenser apertures (40 and 20 µm for condenser aperture n. 3 and 4 respectively JEM 2200 FS, and 20 and 10 µm for the JEM 2011) and small spot size (~2 nm) are used at all times. The disadvantage of these measures is reduced intensity and contrast in the image. Else, the TEM is operated at a


standard 200 kV. Figure 1. S.siderea coral beam damage HRTEM Sample preparation


Coral specimens To investigate the polyp-mineral interface the soft tissue of the polyp consisting of the calicoblastic ectoderm, mesoglea and epidermis, is removed by immersion in fresh water, followed by mechanical brushing and drying. Besides adult specimen skeletal material of coral larvae is also investigated. For this purpose new coral recruits were cultivated in fi ltered seawater at 390 ppm and 750 ppm atmospheric CO2 at 29°C at the aquaculture facilities, James Cook University, Australia. Corallite cross-sections of approx. 1 x 3 mm in size were cut from the cores perpendicular to the growth direction using a diamond cut-off wheel for TEM analysis (adult specimen only). The cross sections or complete larvae where then immersed in Gatan G1 epoxy resin and allowed to cure at 140°C on a hot-plate for 30 minutes to fi ll up the coral pores increasing the stability of the samples. The samples were manually polished down to approximately 50 µm thickness using diamond lapping pads (30 - 0.1 µm) and a tripod holder. Ultimately a bevel is applied, resulting in a sample edge thickness of approximately 20 µm. A molybdenum TEM slot is attached to the sample to provide structural support.


Focused ion beam milling


FIB lamellae are created on the thin polished sample edge using a FEI nova 200 Dual Beam FIB/ SEM from the Leeds Electron Microscopy and Spectroscopy centre. The lamellae are created by applying the h-bar method: a platinum layer is deposited at the edge of the coral sidewall, perpendicular to the general alignment direction of the acicular crystals related to the targeted COC. A approximately 15 x 30 µm lamella is then excavated with a 30 kV Ga+ beam. Bulk milling is performed at 20 nA after which cleaning cross sections are applied at 5 nA and 0.30 nA. The fi nal polish


Figure 2. Coral FIB preparation- optical and SEM


is performed with a 10 kV beam at an angle of 3º at 50 pA. The lamella is milled down to a 100 nm thin section and no lift out is performed to retain specimen context as well as structural support (Figure 2).


Scanning Electron Microscopy (SEM) Instrumentation


In this study a FEI Sirion S-FEG FESEM (fi eld emission SEM) was used from the York Jeol Nanocentre. This instrument is coupled with a Noran EDX system which uses an Oxford INCA analysis system and a 30 mm 2 light element Silicon-Lithium (SiLi) detector.


INTERNATIONAL LABMATE - APRIL 2014


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