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A multiscale approach combining phase-contrast X-ray micro- and nanotomography is applied for imaging a Cretaceous fossil inflorescence in the resolution range from 0.75 μm to 50 nm. The wide range of scale views provides three-dimensional reconstructions from the external gross morphology of the inflorescence fragment to the finest exine sculptures of in situ pollen. This approach enables most of the characteristics usually observed under light microscopy, or with low magnification under scanning and transmission electron microscopy, to be obtained nondestructively. In contrast to previous tomography studies of fossil and extant flowers that used resolutions down to the micron range, we used voxels with a 50 nm side in local tomography scans. This high level of resolution enables systematic affinities of fossil flowers to be established without breaking or slicing specimens.
A significant portion of Mesozoic amber is fully opaque. Biological inclusions in such amber are invisible even after polishing, leading to potential bias in paleoecological and phylogenetic studies. Until now, studies using conventional X-ray microtomography focused on translucent or semi-opaque amber. In these cases, organisms of interest were visualized prior to X-ray analyses. It was recently demonstrated that propagation phase contrast X-ray synchrotron imaging techniques are powerful tools to access invisible inclusions in fully opaque amber. Here we describe an optimized synchrotron microradiographic protocol that allowed us to investigate efficiently and rapidly large amounts of opaque amber pieces from Charentes (southwestern France). Amber pieces were imaged with microradiography after immersion in water, which optimizes the visibility of inclusions. Determination is not accurate enough to allow precise phylogenetic studies, but provides preliminary data on biodiversity and ecotypes distribution; phase contrast microtomography remains necessary for precise determination. Because the organisms are generally much smaller than the amber pieces, we optimized local microtomography by using a continuous acquisition mode (sample moving during projection integration). As tomographic investigation of all inclusions is not practical, we suggest the use of a synchrotron for a microradiographic survey of opaque amber, coupled with microtomographic investigations of the most valuable organisms.
The key strength of hard x-ray full-field microscopy is the large penetration depth of hard x-rays into matter, which allows one to image the interior of opaque objects. Combined with tomographic techniques, the three-dimensional inner structure of an object can be reconstructed without the need for difficult and destructive sample preparation. Projection microscopy and microtomography are now routinely available at synchrotron radiation sources. The resolution of these techniques is limited by that of the detector to 1 µm or slightly less. X-ray images and tomograms at higher spatial resolution can be obtained by x-ray optical magnification, for example, by using parabolic refractive x-ray lenses as a magnifying optic. Combining magnifying x-ray imaging with tomography allows one to reconstruct the three-dimensional structure of an object, such as a microprocessor chip, with resolution well below 1 µm. In x-ray scanning microscopy, the sample is scanned through a small-diameter beam. The great advantage of scanning microscopy is that x-ray analytical techniques such as fluorescence analysis, diffraction, and absorption spectroscopy can be used as contrast mechanisms in the microscope. In combination with tomography, fluorescence analysis makes it possible to reconstruct the distribution of different chemical elements inside an object (fluorescence microtomography), while combining absorption spectroscopy with tomography yields the distribution of different oxidation states of atomic species.
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