Large-Field Electron Imaging and X-ray Elemental ... - ACS Publications

Subscriber access provided by CMU Libraries - http://library.cmich.edu

Article

Large-Field Electron Imaging and X-ray Elemental Mapping Unveil Morphology, Structure and Fractal Features of a Cretaceous Fossil at the Centimetre Scale Naiara C. Oliveira, João H. Silva, Olga A. Barros, Allysson P. Pinheiro, William Santana, Antonio A. F. Saraiva, Odair Pastor Ferreira, Paulo T. C. Freire, and Amauri Jardim Paula Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02815 • Publication Date (Web): 06 Sep 2015 Downloaded from http://pubs.acs.org on September 12, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Large-Field Electron Imaging and X-ray Elemental Mapping Unveil Morphology, Structure and Fractal Features of a Cretaceous Fossil at the Centimetre Scale

Naiara C. Oliveira1, João H. Silva2, Olga A. Barros3, Allysson P. Pinheiro4, William Santana5, Antônio A. F. Saraiva3, Odair P. Ferreira6, Paulo T. C. Freire7 & Amauri J. Paula1* 1

Solid-Biological Interface Group (SolBIN), Departamento de Física, Universidade

Federal do Ceará, P.O. Box 6030, 60455-900, Fortaleza–CE, Brazil 2

Universidade Federal do Cariri, Cidade Universitária, Juazeiro do Norte–CE, Brazil

3

Laboratory of Paleontology, Departamento de Ciências Físicas e Biológicas,

Universidade Regional do Cariri, Crato–CE, Brazil 4

Semiarid Crustaceans Laboratory, LACRUSE, Universidade Regional do Cariri,

Crato–CE, Brazil 5

Sistematic Zoology Laboratory–LSZ, Pró-Reitoria de Pesquisa e Pós–Graduação,

Universidade Sagrado Coração–USC, Bauru–SP, Brazil 6

Laboratory of Advanced-Functional Materials (LaMFA), Departamento de Física,

Universidade Federal do Ceará, Fortaleza–CE, Brazil 7

Laboratory of Raman Spectroscopy, Departamento de Física, Universidade Federal do

Ceará, Fortaleza–CE, Brazil

Corresponding Author * Tel.: +55 85 3366 9270; email: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

ABSTRACT We used here a scanning electron microscopy (SEM) approach that detected backscattered electrons (BSE) and X-rays (from ionisation processes) along a large-field (LF) scan, applied on a Cretaceous fossil of a shrimp (area ~280 mm2) from Araripe Sedimentary Basin. High-definition LF images from BSE and X-rays were essentially generated by assembling thousands of magnified images that covered the whole area of the fossil, thus unveiling morphological and compositional aspects at length scales from micrometres to centimetres. Morphological features of the shrimp such as pleopods, pereopods and antenna located at near-surface layers (undetected by photography techniques) were unveiled in detail by LF BSE images and in calcium and phosphorus elemental maps (mineralised as hydroxyapatite). LF elemental maps for zinc and sulphur indicated a rare fossilisation event observed for the first time in fossils from Araripe Sedimentary Basin: the mineralisation of zinc sulphide (ZnS) interfacing to hydroxyapatite in the fossil. Finally, a dimensional analysis of the phosphorous map led to an important finding: the existence of a fractal characteristic (D=1.63) for the hydroxyapatite-matrix interface, a result of physical-geological events occurring with spatial scale invariance on the specimen, over millions of years.

Keywords: fractal, fossils, scanning electron microscopy, large field scanning, zinc sulphide, X-ray energy dispersive spectroscopy


Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Fossils are remains or vestiges of organisms that lived millions of years ago, formed through several chemical processes, many of them not perfectly understood. They are the result of a process that starts when an organism or its body parts are trapped in sediments in an environment poor in bacteria and rich in minerals dissolved in fluvial flood or oceanic water. They also represent extremely important sources of information on both the environment (e.g., soil, diagenesis of deposits, weather, food availability) and the organisms (e.g., species, territorial distribution, food supply, evolutionary characteristics) from the Earth of ancient times1–5. The correct identification of the fossil material is essential not only for taxonomic studies, but also for studies on paleoecological investigations, population dynamics, biogeography and evolution. However, the lack of diagnostic characteristics due to the commonly poor state of preservation of the material can undermine the precise identification of the organisms. New techniques for detailed analysis of fossils have been increasingly used and progressively contributed to the advance of these studies. Such techniques involve the use of computed tomography scan2,6–8, synchrotron radiation9–12 and several microscopy approaches such as transmission electron microscopy13–15, scanning electron microscopy16–19, infrared-light microscopy20,21, scanning transmission X-ray microscopy22,23 and X-ray fluorescence microscopy24–27. More recently, scanning electron microscopy (SEM) was applied to fossils with areas up to ~230 mm2 using assemblies of magnified electron images and X-ray elemental maps18,19. This bottom-up assembly method was used to generate large, high-definition micrographs that provided detailed morphological information on Cambrian arthropods. Here, we extend this analytical approach to a large-field scan which is capable of unveiling in high definition, and at length scales from micrometres to centimetres, morphological, compositional,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

structural and dimensional aspects at the near-surface layers of a shrimp fossil (area of about 280 mm2) from the Cretaceous period from the Ipubi Formation28. This geological formation belongs to the Araripe Basin29,30, which is located at the geographic borders of Ceará, Piauí and Pernambuco states in Brazil and extends to about 10,000 km2. Araripe Basin has a variety of fossils from the Cretaceous period, including fishes, ostracods, gastropods, turtles, crocodilians, pterosaurs and dinosaurs. It presents a diversified stratigraphic structure consisting of limestones, sandstones, evaporites, shales and concretions. Furthermore, as it holds pyrite, gypsum and calcium carbonate in different geological deposits, it is possible to find fossils produced by diverse chemical processes31. As a consequence, from a paleontological perspective, the Araripe Basin is considered an important geological formation not only because of the diversity and quality of specimens found, but also because of the occurrence of several fossilisation processes32–34. The large-field imaging approach used here for the shrimp fossil comprises the assembly of thousands of magnified images obtained in the SEM from the detection of both electrons and X-rays emitted after the interaction between the incident electron beam and the sample. LF images acquired through this method contain a huge amount of information at several length scales, from a few tens of microns to centimetres, which allows the interpretation of the fossil morphology and the geological context. The method also provides insights regarding the mineralisation of the fossil through the elemental and phase compositions and its fractal analysis, the latter being a powerful tool in order to quantitatively estimate the complex interface growth phenomenon occurred during the fossilisation process.


Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION The large-field scan was carried out in the electron microscope Quanta-450 (FEI) with a field-emission gun (FEG), a 100 mm stage and an X-ray detector (model 150, Oxford) for energy-dispersive X-ray spectroscopy (EDS). The fossil material was inserted in the microscope chamber without a sample preparation. The analyses were performed in a low vacuum (approximately 100 Pa in water vapour) in order to avoid sample charging. Images were acquired at beam acceleration voltages that varied from 5 to 20 kV and with a condenser aperture of 50 µm. For 20 kV of acceleration, which was the best condition for acquiring the BSE images and elemental maps (see Supporting Information for details), the beam current over the specimen was about 45 nA (value provided by the manufacturer considering the conditions used in the column: condenser lens aperture, condenser lens convergence angle and accelerating voltage). A backscattering electron detector (BSE) was fixed at the end of the polar piece and an Xray detector was inserted at a collection angle of 55º with the column axis, positioned approximately at the end of the polar piece. In all analyses, the working distance was set at 15 mm in order to maximise the depth of focus, considering the sample roughness at a micrometre scale. For increasing the beam’s high-vacuum path and minimising spurious beam skirting, a gaseous analytical cone (attached to the BSE detector) was used in all scans. To generate the large-field images, an overlapping of marginal areas (a border which contains 20% of the image area) of adjacent images acquired independently after dislocations of the microscope stage along the x and y axes were performed. The largest constructive interference between the two-dimensional distributions of greyscale values for the overlapped adjacent images determines the best positions for their placement. The resulting images presented in this paper are assemblies of more than 3,600 adjacent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 28

images acquired in individual scans at 1000x magnification (horizontal and vertical fields of 0.41 and 0.29 mm, respectively; 512x368 pixels), and have a definition of 5906x2119 pixels (600 pixel inches–1), with a pixel size corresponding to ~5 µm of the sample. The determination of white pixels corresponding to the interface of the fossil (i.e., hydroxyapatite interface determined in the phosphorus elemental map) was performed by using the integral image method35 programmed on Wolfram Mathematica (see Supporting Information for details). After determining the presence of white pixels inside each box with an increasing lateral size, the fractal dimension (D, or Hausdorff dimension) was calculated. For detailed information regarding the methods used for the LF scan, the image processing and the dimensional analysis, see the Supporting Information.

RESULTS AND DISCUSSION Morphological Assessment of the Fossil through LF BSE Images The fossil material studied here has a size of about 28 mm in length and 10 mm in width (see Figure 1a). Analyses of the material found in the shales of the Ipubi Formation by light microscopy suggested, from a morphological perspective, that it consists of a shrimp (decapod crustacean). Clear morphological details that could enable accurate classification could not be observed through this type of analysis, and thus we generically name the species here as A1 (Figure 1a).


Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1: (a) Photograph of the fossil material from the shales of the Ipubi Formation, Araripe Sedimentary Basin (Ceará, Brazil). (b) Scheme showing the ideal overlapping of greyscale value distributions for adjacent micrographs that were used to generate large-field (LF) images. Large-field scanning electron microscopy (LF–SEM) of the fossil; (c) Backscattered electron (BSE) image assembly with topographic information (BSE T); (d) BSE image assembly with composition information (BSE Z) in low contrast (LC) mode; and (e) BSE image assembly with composition information (BSE Z) obtained in high contrast (HC) mode. Black scale bar corresponds to 10 mm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

In order to image the whole fossil area in high definition, a large-field scan was performed in which the microscope acquires thousands of images with a suitable magnification for all positions covering the fossil area. Although commercial instruments currently available possess a high-precision positioning of the stage, images acquired during this large-field scan present a positioning mismatch from approximately 1 to 10% that prevents a coherent assembling of large-field (LF) images. In this way, this critical process of image assembly can be automated through an image overlapping algorithm that performs an appropriate matching between a pair of images in regard to their two-dimensional distributions of greyscale values (which vary from 0 to 1 at each point of the image plane; see Figure 1b). Through this method, we were able to assemble more than 3,600 micrographs (with horizontal and vertical fields of 0.41 and 0.29 mm, respectively; 512x368 pixels each), covering the whole area of the fossil. The final assembled images (5906x2119 pixels; 600 pixel inches–1) cover a sample area of approximately 280 mm2 and have a pixel size corresponding to ~5 µm of the sample. The minimum spatial resolution in the LF images was ~25 µm, which was the smallest length scale used in the dimensional analysis. Along the large-field scan, two signals manifested from the electron beam-sample interaction were captured: one from backscattered electrons (BSE) and another from Xrays emitted. The signal from BSE was firstly manipulated in order to provide a contrast function related to the topography of the sample (BSE T), which was generated by subtracting the signal captured at a half of the BSE annular detector from the signal captured at the other half (see Figure 1c). By summing both signals (for the full annulus), a contrast function related to the composition of the sample (BSE Z) was acquired, originating from the relationship between the BSE yield and the atomic mass (g mol‒1) of each element present in the sample (see Figures 1d and 1e). These different


Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contrast functions used for topography (BSE T) and composition (BSE Z) can be compared through histograms of greyscale values extracted from the images (see right panels in Figures 1c, 1d and 1e). The topographic contrast (BSE T, see right panel of Figure 1c) along the image does not substantially vary, as the fossil surface is rather flat, thus resulting in a greyscale histogram that has a progressive decrease in the number of pixels from black (zero intensity) to grey tones (around 0.6). In addition, for images associated with BSE Z (see Figures 1d and 1e), the contrast is rather different and can also be altered in order to obtain very distinct information regarding either the morphology or the composition of the shrimp. Low contrast LF BSE images (BSE Z LC, see Figure 1d) are suitable for providing differentiations in the chemical composition along the fossil sample but fail to fully reveal the fossil morphological contours. These low contrast images present a greyscale histogram with a larger quantity of dark pixels (approximately 1 x 107 black pixels; see right panel in Figure 1d). Regions with tones close to white (i.e., greyscale value = 1) reveal areas of the fossil that have different composition, and regions in grey tones partially reveal the fossil morphology imprinted in the matrix. However, as these samples commonly comprise a large quantity of elements, sixteen in this particular case, the interpretation of low contrast BSE Z images becomes difficult without the association with elemental maps. These maps were obtained through the X-rays emitted from atomic ionisation processes occurring through the electron beam interaction. As further discussed here, the brighter tones in Figure 1d are mainly associated with the presence of zinc (Zn), the heaviest (highest atomic mass) among the major elements present in the fossil, with the highest BSE yield. On the other hand, in high contrast BSE images (BSE Z HC; see Figure 1e), the quantity of brighter tones in the histograms was increased, resulting in a balance of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

black and white pixels along the image (i.e., ~3 x 106 pixels for both; see right panel in Figure 1e). In these conditions, it is possible to fully reveal the fossil contours due to the BSE yield of calcium (Ca). However, information regarding the composition along the fossil is lost by the contrast increase. In this high contrast image, the pleopods and pereopods of the shrimp, features that are undetected in Figure 1a, were revealed in near-surface layers. Compositional Assessment through LF Elemental Maps The X-rays emitted from the specimen were detected during the LF scan along with the detection of backscattered electrons, thus allowing the assembly of the elemental maps. Considering the effect of the acceleration voltage on the large-field elemental maps produced, it was observed that scans with the electron beam accelerated at 5 kV produced images with a higher noise-to-signal ratio when compared to the images obtained at 20 kV for the same beam dwell time (60 µs per pixel). In addition, as no significant morphological differences were observed when comparing the maps generated at 5 and 20 kV up to a scale of a few tens of microns (see Figure S2 in the Supporting Information), elemental maps obtained at 20 kV were preferably used due to the low noise-to-signal ratio (for detailed considerations on the use of this accelerating voltage, see the Supporting Information). The cumulative EDS spectrum containing the sum of all spectra (more than 3,600) obtained through the LF scan is represented at the top panel of Figure 2. Maps for elements present in the specimen with a relative concentration above 1.0 wt% are represented at the bottom panels of the same figure. These maps were rendered by filtering the X-ray signal through an energy gap of 80 eV (spectral resolution = ~5 eV), centred at the energy associated with the peak maximum intensity for each element in the EDS spectrum (see Figure 2, top panel). Maps for elements with relative concentration above 1.0 wt% were generated using Kα transitions


Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(i.e., electronic transitions from L to K shells), which represent the most accurate signal that could be used for all elements. Although the carbon signal was excluded in maps and calculations due to the possible influence of contamination during the fossil handling, its exclusion did not impact the quantitative interpretations performed here, as they were carried out by using just ratios between the elements’ relative molar concentrations (in molar units). In the fossil, carbon is associated mainly as carbonates (e.g. CaCO3).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

Figure 2: Top panel: total energy-dispersive X-ray spectrum (EDS) for the whole fossil area. Bottom panels: elemental maps generated by using the signal of each ionisation peak described. Elemental maps are represented from left to right, top to bottom, as a function of their relative concentrations (wt%) calculated from the total energydispersive X-ray spectrum. Elements with quantities larger than 1.0 wt% are shown. White scale bar corresponds to 10 mm. 12 ACS Paragon Plus Environment

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fossils previously studied from the Ipubi Formation36 were mainly imprints between dark shales that are rich in organic matter and calcium sulphates. When phosphorus (P) and calcium (Ca) are traced in the elemental maps (mineralised as hydroxyapatite [Ca5(PO4)3OH], see Figure 2), it is possible to observe in better detail characteristics of the abdomen, the partially preserved carapace, parts of the pleopods and other cephalic appendages (i.e., antenna and antennule). Maps also make possible the improvement of the description of other characteristics such as the thoracic appendages (pereopods 3-5), the abdominal pleura of somites 4-5, and new morphological details of the pleopods, uropods and telson. The rostral spines were not preserved, and parts of the antennas and antennules were curved upwards, above the cephalothorax. An appropriate interpretation of the fossil composition is better performed in association with elemental maps (see Figure 3), as there are sixteen elements identified through EDS (see Table S1 and Table S2 in the Supporting Information for details regarding the energy lines used for detection and the elements relative concentrations, respectively). On the elemental composition of the fossil, it was observed that there are three characteristic regions: the matrix (region A) containing mainly oxygen (O), silicon (Si), calcium (Ca), sulphur (S), aluminium (Al), magnesium (Mg) and iron (Fe); a phosphorus-rich region of the fossil (region B) containing oxygen, calcium, phosphorous (P), fluorine (F), sulphur and sodium (Na); and finally a region in the fossil containing mostly sulphur and zinc (Zn) (region C).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

Figure 3: EDS spectra from specific regions of the fossil associated with the areas shown in the LF BSE Z LC image (top panel). Regions defined are (A) matrix, (B) phosphorus-rich area of the fossil and (C) zinc- and sulphur-rich area. Bottom panels: resulting spectra from the sum of at least 5 spectra acquired in each region described.


Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The calculation of the relative concentration for all elements identified in the cumulative EDS spectrum (see Figure 2) as well as in spectra for regions A, B and C (see Figure 3) is represented in Table S2 in the Supporting Information. Data from the cumulative EDS spectrum for the whole area of the fossil are represented in Table S2 in an ascending order in regard to the atomic mass (g mol‒1) of elements detected. Surprisingly, the peaks of trace elements present at relative concentrations smaller than 1.0 wt% could be detected in the cumulative EDS spectrum though their relative concentration (wt%) could not be considered in quantitative interpretations. This is the case of chlorine (Cl), potassium (K), titanium (Ti), cerium (Ce) and ytterbium (Yb). The fluorine map indicates that the presence of this element is limited to the boundaries of the fossil, largely overlapping the phosphorus map. This fact suggests the possibility of a partial substitution of the hydroxyl group of hydroxyapatite by fluorine [Ca5(PO4)3(OH(1‒x)Fx)]. The total substitution can lead to the formation of fluoroapatite [i.e. Ca5(PO4)3F], which can provide to the fossil more chemical stability and resistance to dissolution37,38. Another major conclusion was taken considering the overlapping of zinc and sulphur elemental maps in Figure 2, and also considering their normalised relative molar concentrations (NRMC, in molar units) calculated through spectra from region C (approximate ratio of 97.5 mol of sulphur to 99.8 mol of Zn; see Table S2 in the Supporting Information), which confirms the presence of zinc sulphide (ZnS) in the fossil. The presence of (Zn1‒xFex)S (wurtzite or sphalerite with a high amount of iron ["x" value]) and FeS2 (pyrite) must be considered to a lesser extent, because there is an overlapping of Fe, Zn and S elemental maps inside the fossil contours, but the Fe normalised relative molar concentration is very low in regard to Zn and S (ratio of approximately 1:100; see Table S2). Although the presence of sulphur in other compositions such as sulphates (e.g. CaSO4.xH2O) must be also considered, EDS



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

spectra for this region show an approximate sulphur:oxygen molar ratio (around 2:1) that does not indicate the presence of sulphates in high concentrations, thus supporting the conclusion regarding the larger mineralization of ZnS or (Zn1‒xFex)S, with a low "x" value. Furthermore, the oxygen wt% value also comprises signals from impurities on the fossil surface and from the water vapour used in the chamber (to attain 100 Pa), which leads to the conclusion that sulphur:oxygen molar ratio on Region C is in fact larger than 2:1. Fossilisation through the mineralisation of pyrite in Araripe Sedimentary Basin was previously observed36. However, this is the first observation of the formation of zinc sulphide in fossils from this basin. The presence of ZnS in fossils, appearing as sphalerite or wurtzite, was observed in biogenic remains in the form of internal casts, as infillings of voids in skeletons and also as replacement of skeleton materials39,40. More specifically, this compound has been observed in fossils from deep-sea hot springs at hydrothermal vent fields present along crests of volcanic spreading ridges41–43. Fossils in these areas were formed from fauna commonly comprising worms, crabs and a variety of fishes44,45. The mineralisation of ZnS in these fossils is related to the acidification of the solution and the presence of hydrogen sulphide46, which consequently induce the precipitation of zinc sulphide44,45. The pivotal point, however, is that the presence of sulphur and zinc in fossils is a limited and rare event. In the case of gastropods39, the organisms accumulate a large quantity of Zn during their lives, allowing this chemical element to be available in the environment after their deaths to form compounds such as ZnS. Regarding the shrimp A1, the presence of Zn can also be attributed to an accumulation process during life, although it is known through modern toxicological studies that shrimps are not an efficient trap for Zn47,48 as are gastropods39,49–51, and are able to regulate their total body


Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


levels of Zn52. Obviously, we cannot exclude a different origin for Zn, such as its introduction by water with a high concentration of zinc. The process of ZnS formation itself can be very complex, as pointed out in the case of gastropod fossils. In fact, at least two possibilities were found: a direct infilling of the carbonate shells by ZnS, or the infilling of the carbonate shells by calcite and/or pyrite, with the replacement of these materials by sphalerite or wurtzite39. Related to the direct infilling by ZnS, it was shown that the presence of sulphate-reducing bacteria cultured in a ZnSO4/FeSO4 medium leads to ZnS precipitation, but iron sulphides like pyrite are not precipitated53. This is an important finding that can account for the formation of zinc sulphide in the shrimp fossil studied here because, as our data show, only about 0.5 wt% of Fe was found in the region of the fossil where ZnS is present. On the other hand, one cannot discard a more complex mechanism involving the precipitation of ZnS, calcite and pyrite. The fossilisation process of some fossils from the Ipubi Formation occurs through pyritisation in several stages, as previously shown by our group36. In this way, the hypothesis of formation of ZnS through different stages is another possibility, similarly to the process occurring for gastropods39. Firstly, the animal is infilled by iron sulphides and calcite, and finally sphalerite or wurtzite substitutes the calcite casts.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

Dimensional Analysis of the Fossil Hydroxyapatite Interface Although it was observed in LF images that the fossil was preserved from a macroscopic perspective, thus allowing paleontological and morphological interpretation, at small length scales (microns), the contours of the fossil-matrix interface are irregular and complex, a result of the taphonomic processes. In this context, a suitable approach for obtaining quantitative information about these processes is the dimensional analysis of the fossil interfaces, more specifically the contours that define the interfaces between the fossil and the matrix. This quantitative analysis could be performed at different length scales because LF images possess a contour definition from micrometres to centimetres. The dimension was assessed through a box-counting method that divides the image into a determined number of square areas (or boxes; N(l)) which is dependent on the lateral size (l), thus covering the whole image (for details, see the Methods section and the Supporting Information). As seen in Figures 4a to 4c, the fossil-matrix interface dimension was assessed by determining the presence of white pixels inside these boxes after the phosphorus elemental map was binarised (i.e., converted to a black and white scale, with 0 and 1intensities, respectively) and the contours revealed by applying a differentiation function on the resulting binarised contrast function (i.e., edge-finding process). Considering the approach introduced by Mandelbrot for the dimensional evaluation of natural events54, the edge-finding process used here reveals what would be analogous to “islands and lakes”, the boundaries which correspond to the fossil-matrix interface. Therefore, boundaries (i.e., contours) of individual islands and lakes do not contact each other. The hydroxyapatite interface was analysed through the boundaries revealed in the phosphorus elemental map. The dimensional analysis for the P elemental map (see Figure 4d) shows the existence of a linear relationship between the logarithms 18 ACS Paragon Plus Environment

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the number of boxes used to cover the two-dimensional phosphorus interface (log[N(l)]) and the inverse of the box size (log[1/l]), confirming the fractal spatial arrangement of this interface, a result related to the hydroxyapatite mineralisation process. The linear fit precisely provided a fractal dimension D (Hausdorff dimension) of 1.63 for the interface. The fractal dimension in this case demonstrates the spatial scale invariance and self-similarity of the hydroxyapatite-matrix interface, and it is a number specifically related to the fossil contours as well as to its fragmentation attained over time, both ruled by diffusion phenomena that occurred along and across the P elemental map plane analysed (see Figure 4a). An illustrative comparison can be made with the dimensional features of continents and islands. The fractal dimension of the Australian coastline is 1.1355. This number rises if (i) the continent is fragmented in the sea (Norway coastline has D=1.52); and/or if (ii) the contours of the lakes and rivers inside the continent are considered along with the contours of the coastline (i.e., water-crust interface seen from above). More specifically considering fossils, values of D close to this obtained (1.63) were observed for coral corallites from the Upper Jurassic56, graphoglyptid trace fossils57,58, and rangeomorph fronds from the Ediacaran Period59. However, it must be mentioned that similar fractal dimensions can correspond to radically different morphologies, so that other morphometric analyses must be performed in order to interpret them. Finally, the results from the box-counting method are influenced by the definition of the images used. In this way, imaging methods such as this here presented, which allows the obtainment of images in ultra-high definition, can substantially improve the precision of the calculation of the fractal dimension. From a macroscopic perspective, the morphology of the living animal was distributed along the space (Euclidean) through an auto-organised arrangement as a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

result of emergent properties from its intrinsic complex nature. As observed in the LF images, its outer morphological contours had macroscopic dimensional characteristics that are partially preserved in the fossil. In addition, at the micrometre scale, the fossilisation of the animal is a result of long-term physical-geological processes that took place correlated with the morphology of the living animal: minerals nucleation and growth were limited to some extent by the animal contours. In a first analysis, it must be considered that geometrically well-defined mathematical models can generate fractals with fragmentation and dimension close to the one observed for this fossil (for instance, fractals based on generalized Koch curves can attain D~1.61)54. However, the hydroxyapatite-matrix interface was formed through mineralisation events that can be dimensionally best correlated with randomly generated fractals (e.g., Brown fractals)60.


Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4: (a) Phosphorus elemental map from Κα electronic transition associated with the presence of hydroxyapatite. (b) Example of the binarisation and edge-finding processes sequentially applied to a determined area of the phosphorus map. (c) Hydroxyapatite-matrix interface revealed by imaging processing performed for the whole phosphorus map. (d) Plot of the logarithms of the number of boxes used to cover the two-dimensional hydroxyapatite interface (log[N(l)]) and the inverse of the box size (log[1/l]) found in the dimensional analysis. Linear fitting of the points is showed as an inset.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

CONCLUSIONS A large-field (LF) scanning electron microscopy approach was used here for analysing a Cretaceous fossil of a shrimp from Araripe Sedimentary Basin (Brazil) by imaging through the detection of backscattered electrons (BSE) and X-rays from ionisation processes. The resulting LF images revealed morphological features of the fossil at near-surface layers, which could not be detected through light microscopy. The morphological details of the pleopods, pereopods, antennae, antennules and somites of the shrimp were better visualised in the LF BSE images and also in LF calcium and phosphorus elemental maps, which are present in the fossil mineralised as hydroxyapatite. Furthermore, the correlation of LF elemental maps with EDS spectra of specific regions of the fossil revealed important aspects of the fossilisation process. In this particular case, it was observed here that along with the fossilisation of the animal through the formation of hydroxyapatite; zinc sulphide (ZnS) was also formed, interfacing with the hydroxyapatite. This is a rather rare process of fossilisation, for the first time observed in the Araripe Sedimentary Basin. As the LF images possess precise information at several length scales, ranging from a few tens of microns to centimetres, this imaging approach also allowed a dimensional analysis of the specimen, more specifically of the interface formed between hydroxyapatite and the matrix. This numerical analysis surprisingly revealed the existence of a fractal arrangement of this interface (Hausdorff dimension of 1.63), a result of physical-geological events that occurred over millions of years, with spatial scale invariance. Finally, it must be also mentioned that this image processing approach can be extended to other scientific contexts, for instance, to polymers, metals, rocks and organic tissues, in which morphological, structural, compositional and dimensional characterisation must be


Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


carried out along a wide range of length scales, thus providing unique information for each context.

ACKNOWLEDGEMENTS The authors gratefully thank Central Analítica–UFC/CT–INFRA/MCTI– SISNANO/Pró-Equipamentos CAPES for providing the FEG–SEM, as well as CNPq and FUNCAP for funding this research (Grant no. 446800/2014-7).

SUPPORTING INFORMATION Detailed information on the methods used, scanning electron micrographs and X-ray energy-dispersive spectroscopy tables. This material is available free of charge via the Internet at http://pubs.acs.org.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

FOR TABLE OF CONTENTS ONLY


Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES

(1)

Thomas, D. B.; Nascimbene, P. C.; Dove, C. J.; Grimaldi, D. A.; James, H. F. Sci. Rep. 2014, 4, 5226.

(2)

Castanhinha, R.; Araújo, R.; Júnior, L. C.; Angielczyk, K. D.; Martins, G. G.; Martins, R. M. S.; Chaouiya, C.; Beckmann, F.; Wilde, F. PLoS One 2013, 8, e80974.

(3)

Zabini, C.; Schiffbauer, J. D.; Xiao, S.; Kowalewski, M. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2012, 326-328, 118–127.

(4)

Bennett, C. E.; Williams, M.; Leng, M. J.; Siveter, D. J.; Davies, S. J.; Sloane, H. J.; Wilkinson, I. P. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2011, 305, 150– 161.

(5)

Maisey, J. G. Environ. Biol. Fishes 1994, 40, 1–22.

(6)

Ketcham, R. A.; Carlson, W. D. Comput. Geosci. 2001, 27, 381–400.

(7)

Spoor, F.; Wood, B.; Zonneveld, F. Nature 1994, 369, 645–648.

(8)

Sereno, P. C.; Wilson, J. a.; Witmer, L. M.; Whitlock, J. a.; Maga, A.; Ide, O.; Rowe, T. a. PLoS One 2007, 2, e1230.

(9)

Houssaye, A.; Xu, F.; Helfen, L.; Buffrénil, V. De; Baumbach, T.; Tafforeau, P. J. Vertebr. Paleontol. 2011, 31, 2–7.

(10)

Bergmann, U.; Morton, R. W.; Manning, P. L.; Sellers, W. I.; Farrar, S.; Huntley, K. G.; Wogelius, R. a; Larson, P. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 9060–9065.

(11)

Muscente, A. D.; Marc Michel, F.; Dale, J. G.; Xiao, S. Precambrian Res. 2015, 263, 142–156.

(12)

Anné, J.; Edwards, N. P.; Wogelius, R. A.; Tumarkin-Deratzian, A. R.; Sellers, W. I.; van Veelen, A.; Bergmann, U.; Sokaras, D.; Alonso-Mori, R.; Ignatyev, K.; Egerton, V. M.; Manning, P. L. J. R. Soc. Interface 2014, 11, 20140277.

(13)

Javaux, E. J.; Knoll, A. H.; Walter, M. R. Geobiology 2004, 2, 121–132.

(14)

Li, J.; Bernard, S.; Benzerara, K.; Beyssac, O.; Allard, T.; Cosmidis, J.; Moussou, J. Earth Planet. Sci. Lett. 2014, 400, 113–122.

(15)

Brasier, M. D.; Antcliffe, J.; Saunders, M.; Wacey, D. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 4859–4864.

(16)

Garcia-Ruiz, J. M.; Hyde, S. T.; Carnerup, A. M.; Christy, A. G.; Van Kranendonk, M. J.; Welham, N. J. Science 2003, 302, 1194–1197. 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

(17)

Matzke-karasz, R.; Neil, J. V.; Smith, R. J.; Symonová, R.; Morkovský, L.; Archer, M.; Hand, S. J.; Cloetens, P.; Tafforeau, P. Proc. R. Soc. B Biol. Sci. 2014, 281, 20140394.

(18)

Ma, X.; Cong, P.; Hou, X.; Edgecombe, G. D.; Strausfeld, N. J. Nat. Commun. 2014, 5, 3560.

(19)

Ma, X.; Hou, X.; Edgecombe, G. D.; Strausfeld, N. J. Nature 2012, 490, 258– 261.

(20)

Riquelme, F.; Northrup, P.; Ruvalcaba-Sil, J. L.; Stojanoff, V.; Peter Siddons, D.; Alvarado-Ortega, J. Appl. Phys. A 2014, 116, 97–109.

(21)

Edwards, N. P.; Barden, H. E.; van Dongen, B. E.; Manning, P. L.; Larson, P. L.; Bergmann, U.; Sellers, W. I.; Wogelius, R. a. Proc. R. Soc. B Biol. Sci. 2011, 278, 3209–3218.

(22)

Benzerara, K.; Yoon, T. H.; Tyliszczak, T.; Constantz, B.; Spormann, A. M.; Brown, G. E. Geobiology 2004, 2, 249–259.

(23)

Cosmidis, J.; Benzerara, K.; Gheerbrant, E.; Estève, I.; Bouya, B.; Amaghzaz, M. Geobiology 2013, 11, 139–153.

(24)

Schweitzer, M. H.; Zheng, W.; Cleland, T. P.; Goodwin, M. B.; Boatman, E.; Theil, E.; Marcus, M. A.; Fakra, S. C. Proc. R. Soc. B Biol. Sci. 2014, 281, 20132741.

(25)

Edwards, N. P.; Manning, P. L.; Bergmann, U.; Larson, P. L.; van Dongen, B. E.; Sellers, W. I.; Webb, S. M.; Sokaras, D.; Alonso-Mori, R.; Ignatyev, K.; Barden, H. E.; van Veelen, A.; Anné, J.; Egerton, V. M.; Wogelius, R. a. Metallomics 2014, 6, 774–782.

(26)

Ryan, C. G.; Siddons, D. P.; Kirkham, R.; Li, Z. Y.; de Jonge, M. D.; Paterson, D. J.; Kuczewski, A.; Howard, D. J.; Dunn, P. A.; Falkenberg, G.; Boesenberg, U.; de Geronimo, G.; Fisher, L. A.; Halfpenny, A.; Lintern, M. J.; Lombi, E.; Dyl, K. A.; Jensen, M.; Moorhead, G. F.; Cleverley, J. S.; Hough, R. M.; Godel, B.; Barnes, S. J.; James, S. A.; Spiers, K. M.; Alfeld, M.; Wellenreuther, G.; Vukmanovic, Z.; Borg, S. J. Phys. Conf. Ser. 2014, 499, 012002–012012.

(27)

Wogelius, R. A.; Manning, P. L.; Barden, H. E.; Edwards, N. P.; Webb, S. M.; Sellers, W. I.; Taylor, K. G.; Larson, P. L.; Dodson, P.; You, H.; Da-qing, L.; Bergmann, U. Science 2011, 333, 1622–1626.

(28)

Da Silva, J. H.; de Sousa Filho, F. E.; Saraiva, A. Á. F.; Andrade, N. A.; Viana, B. C.; Sayão, J. M.; Abagaro, B. T. D. O.; Freire, P. D. T. C.; Saraiva, G. D. J. Spectrosc. 2013, 2013, 1–7.

(29)

Antonietto, L. S.; Gobbo, S. R.; Do Carmo, D. A.; Assine, M. L.; Fernandes, M. A. M. C. C.; Lima e Silva, J. E. J. Paleontol. 2012, 86, 659–668.


Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30)

Martill, D. M. Cretac. Res. 2007, 28, 895–920.

(31)

Freire, P. T. C.; Silva, J. H.; Sousa-Filho, F. E.; Abagaro, B. T. O.; Viana, B. C.; Saraiva, G. D.; Batista, T. A.; Barros, O. A.; Saraiva, A. A. F. J. Raman Spectrosc. 2014, 45, 1225–1229.

(32)

Pinheiro, A. P.; Saraiva, A. Á. F.; Santana, W. An. Acad. Bras. Cienc. 2014, 86, 663–670.

(33)

Fara, E.; Saraiva, A. A. F.; Almeida Campos, D.; Moreira, J. K. R.; Carvalho Siebra, D.; Kellner, A. W. A. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2005, 218, 145–160.

(34)

Menon, F.; Makarkin, V. N. Palaeontology 2008, 51, 149–162.

(35)

Viola, P.; Jones, M. Proc. 2001 IEEE Comput. Soc. Conf. Comput. Vis. Pattern Recognition. CVPR 2001 2001, 1, I511–I518.

(36)

Freire, P. T. C.; Abagaro, B. T. O.; Souza Filho, F. E.; Silva, J. H.; Saraiva, A. A. F.; Brito, D. D. S.; Viana, B. C. In Pyrite: Synthesis, Characterization and Uses; Whitley, N., Vinsen, P. T., Eds.; Nova Science Publishers, 2013; pp 123–140.

(37)

Snioszek, M.; Telesinski, A.; Musik, D.; Zakrzewska, H. J. Elem. 2008, 13, 675– 684.

(38)

Grynpas, M. D. J. Bone Miner. Res. 1990, 5, 169–175.

(39)

Szczepanik, P.; Sawlowicz, Z. Naturwissenschaften 2008, 95, 869–876.

(40)

Zbinden, M.; Martinez, I.; Guyot, F.; Cambon-Bonavita, M.-A.; Gaill, F. Eur. J. Mineral. 2001, 13, 653–658.

(41)

Halliday, I.; Blackwell, A. T.; Griffin, A. A. Science 1984, 223, 1407–1409.

(42)

Grassle, J. F. Science 1985, 229, 713–717.

(43)

Jannasch, H. W.; Mottl, M. J. Science 1985, 229, 717–725.

(44)

Corliss, J. B.; Dymond, J.; Gordon, L. I.; Edmond, J. M.; Herzen, R. P. von; Ballard, R. D.; Green, K.; Williams, D.; Bainbridge, A.; Crane, K.; Andel, T. H. van. Science 1979, 203, 1073–1083.

(45)

Spiess, F. N.; Macdonald, K. C.; Atwater, T.; Ballard, R.; Carranza, A.; Cordoba, D.; Cox, C.; Diaz Garcia, V. M.; Francheteau, J.; Guerrero, J.; Hawkins, J.; Haymond, R.; Hessler, R.; Juteau, T.; Kastner, M.; Larson, R.; Luyendyk, B.; Macdougall, J. D.; Miller, S.; Normark, W.; Orcutt, J.; Rangin, C. Science 1980, 207, 1421–1433.

(46)

Edmond, J. M.; Von Damm, K. L.; Mcduff, R. E.; Measures, C. I. Nature 1982, 297, 187–191. 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

(47)

Ahsanullah, M.; Negilski, D. S.; Mobley, M. C. Mar. Biol. 1981, 64, 299–304.

(48)

Nunez-Nogueira, G.; Rainbow, P. S.; Smith, B. D. J. Exp. Mar. Bio. Ecol. 2006, 332, 75–83.

(49)

Roméo, M.; Gharbi-Bouraoui, S.; Gnassia-Barelli, M.; Dellali, M.; Aïssa, P. Sci. Total Environ. 2006, 359, 135–144.

(50)

Kulikova, N. N.; Maksimova, N. V.; Suturin, A. N.; Paradina, L. F.; Sitnikova, T. Y.; Timoshkin, O. A.; Saibatalova, E. V.; Khanaev, I. V. Geochemistry Int. 2007, 45, 478–489.

(51)

Gundacker, C. Environ. Pollut. 2000, 110, 61–71.

(52)

White, S. L.; Rainbow, P. S. Mar. Ecol. Prog. Ser. 1982, 8, 95–101.

(53)

Labrenz, M.; Druschel, G. K.; Thomsen-Ebert, T.; Gilbert, B.; Welch, S. A.; Kemner, K. M.; Logan, G. A.; Summons, R. E.; De Stasio, G.; Bond, P. L.; Lai, B.; Kelly, S. D.; Banfield, J. F. Science 2000, 290, 1744–1747.

(54)

Mandelbrot, B. B. The fractal geometry of nature, Third.; Freeman: New York, 1983.

(55)

Mandelbrot, B. Science 1967, 156, 636–638.

(56)

Martin-Garin, B.; Lathuilière, B.; Verrecchia, E. P.; Geister, J. Coral Reefs 2007, 26, 541–550.

(57)

Lehane, J. R.; Ekdale, A. A. Palaios 2013, 28, 23–32.

(58)

Lehane, J. R.; Ekdale, A. A. J. Paleontol. 2014, 88, 747–759.

(59)

Cuthill, J. F. H.; Morris, S. C. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13122– 13126.

(60)

Mandelbrot, B. B. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 3825–3828.


Large-Field Electron Imaging and X-ray Elemental ... - ACS Publications

Recommend Documents