73 Sample preparation
Specimen preparation for SEM is usually much quicker and less involved as compared to TEM specimen preparation as the samples need not be electron transparent. Since there is a limit to the depth of field and we are interested in the cross section perpendicular to the growth direction of coral skeletons, a bulk cut followed by polishing is performed to a thickness of approximately 100-200 µm using the tripod polishing method, but without applying a bevel. This particular thickness range makes the specimen light transparent, which enables us to combine transmission optical microscope data with surface SEM data.
Figure 3. P.lobata coral SEM series Figure 5. Schematic Diurnal cycle
Specimens are subsequently lightly etched with deionised water (coral larvae) and dilute acetic acid-water mixture (adult coral specimen) to reveal the microstructure (Figure 3).
Summary of Conclusions (as published in recent paper*)
In this study we show that new insights can be gained on the nano- and microstructure of corallites by TEM investigation of large-scale (15 x 30 µm) FIB lamellae from adult and juvenile scleractinian coral skeletal specimens. By leaving the FIB prepared lamella within the coral skeletal context (no lift out) the lamella is mechanically more stable and durable while being directly comparable to the larger scale (several tens of microns) not ion-milled skeletal areas by optical analysis. Thus we could identify a crystallographic evolution from a centre of calcification outward over acicular and granular (daily) bands. In the adult Porites lobata specimen, a succession of randomly orientated nanocrystals with high porosity, followed by partly aligned nanocrystals with high porosity, to dense acicular crystals of several microns in length, was observed. A new layer of nanocrystals followed by the same succession is then repeated, in correlation with the contrast banding seen in transmission light images. Both the partly-aligned nanocrystals and the densely packed acicular bundles are preferentially oriented in the [001] crystallographic growth direction (Pmcn space group notation), (Figure 4).
Figure 4. P.lobata coral SAD series nanocrystals
References [1] D. B. Williams, C. B. Carter, Transmission electron microscopy: a textbook for materials science, Springer, 2009. [2] S. Horiuchi, T. Hanada, M. Ebisawa, et al., ACS nano 3 (2009), pp. 1297-1304.
*Paper: Microstructural evolution and nanoscale crystallography in scleractinian coral spherulites Renée van de Locht et al, Journal of Structural Biology 183 (2013) pp.57-65
We draw a parallel to the diurnal photosynthetic cycle of the zooxanthellae in symbiotic corals that control the levels of oxygen and carbon availabilities, both recognised as significant drivers of coral calcification processes. The transport of large amounts of glycerol by zooxanthellae and its potential impact on coral calcification was also discussed. This process could play a role in the specific alignment of the aragonite crystals as was demonstrated by synthetic experiments with OH-group containing additives by Sand et al. (2011) and the TEM investigation of these precipitates by the authors. The juvenile Acropora millepora specimen also showed the large acicular crystals interrupted by thin porous bands, but lacked the nanocrystalline phase, which may be linked to the absence of zooxanthellae and thus the typical daily cycle. But at this stage a comparison is not possible as we are dealing with different species and growth rates and morphologies are also likely to play an important role (Figure 5).
Finally, we conclude that our data do not support a growth mechanism via self-assembly of submicron-sized units associated with a mesocrystalline morphology, as the nanocrystalline areas are found to be polycrystalline on the micrometer scale with no extended co- orientation. We therefore conclude that the coral mineralisation follows a classical crystallisation pathway.
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www.labmate-online.com/articles RISE Microscopy for Correlative Raman-SEM Imaging Launched at Analytica 2014
Tescan Orsay Holdings and WITec GmbH jointly launched RISE Microscopy at Analytica 2014. RISE Microscopy is a novel correlative microscopy technique which combines confocal Raman Imaging and Scanning Electron (RISE) Microscopy within one integrated microscope system. This unique combination provides clear advantages for the microscope user with regard to comprehensive sample characterisation. The RISE Microscope enables for the first time the acquisition of SEM and Raman images from the same sample area and the correlation of ultra-structural and chemical information with one microscope system.
Both analytical methods are fully integrated into the RISE Microscope. Between the different measurements an extremely precise scan stage automatically transfers the sample inside the microscope’s vacuum chamber and re-positions it. The integrated RISE software carries out the required parameter adjustments and instrument alignments. The acquired results can then be correlated and the Raman and SEM images overlaid. “RISE Microscopy enables unprecedented opportunities for the most comprehensive ultra-structural and molecular sample analyses,” explained Dr Olaf Hollricher, CEO and Director R&D at WITec “The novel RISE Microscope is another striking example of WITec’s enormous innovative strength. It fulfils all requirements of an outstanding, correlative microscopy technique and will convince the Raman as well as the SEM community.”
TESCAN and WITec arranged worldwide sales and after-sales cooperation for the RISE Microscope to take advantage of the synergy effects of both companies.
The RISE Microscope provides all functions and features of a stand-alone SEM and a confocal Raman microscope. Both SEM and Raman are high-resolution imaging techniques with sub-nanometer and diffraction limited 200 - 300 nanometer resolution, respectively. In Raman imaging mode the sample can be scanned through a range of 250 µm x 250 µm x 250 µm. RISE Microscopy pairs ease-of-use with exceptional analysing benefits and is therefore suited to a large variety of applications such as nanotechnology, materials science, and life science.
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