The Apatite Crystal in Fossil Cells with Fluorescence - Crystal Growth

We found fluorescent apatite crystals in the fossil cells with different sizes and shapes. Using energy dispersive spectroscopy and powder X-ray diffr...
1 downloads 0 Views 3MB Size
Download PDF
The Apatite Crystal in Fossil Cells with Fluorescence Huimei Chi,*,†,‡ Man Feng,§ Yuanchang Chen,‡ Chun Ding,‡ Linjuan Gu,‡ Jingwu Ma,‡ and Zuhong Lu*,†,‡,| State Key Laboratory of Bioelectronics, Southeast UniVersity, Nanjing, 210096, China, School of Biomedical and Engineering, Southeast UniVersity, Nanjing, 210096, China, Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences, Nanjing, 210008, China, and Key Laboratory of Child DeVelopment and Learning Science, Southeast UniVersity, Nanjing, 210096, China

CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 2 676–681

ReceiVed September 7, 2007; ReVised Manuscript ReceiVed October 16, 2008

ABSTRACT: The microfossils from Neoproterizoic Doushantuo Formation, Southeast China, include multicellular animal and algae fossils with exquisite cellular or even subcellular structure preserved, which show the beginning of biodiversity in the Precambrian ocean about 600 million years ago. The fossil cells can fluoresce when excited by laser light and have a steady fluorescence emission in the visible spectrum. The organic matter was thought as the fluorescence source. However, our observation of the microfossils from Doushantuo Formation, Weng‘an reveals another fluorescence source. We found by scanning electron microscopy and transmission light microscopy that the fossil cells with fluorescence are composed of small crystals in a naturally self-assembled manner. Through energy dispersive spectroscopy we know that the crystal is mainly composed of Ca, P, O, and F elements. Furthermore, we performed powder X-ray diffraction to confirm that the crystal compound is Ca5(PO4)3F. Therefore, we consider that crystallization of Ca5(PO4)3F is the main reason that causes the fossil cells to illuminate.

1. Introduction The phosphatized microfossils from Doushantuo Formation, Guizhou, China, contain multicellular animal and algae fossils,1 which provide a main window for early life evolution. In recent years, palaeontologists found more and more new lines of evidence of ancient multicellular organisms, including sponges, cnidarians embryos, small bilateral animals, and the earliest lichen-like fossils;2 all the evidence mentioned above indicate biodiversity about 551-632 million years ago.3 However, discussion always existed concerning the interpretation of some fossil specimens. For example, some researchers called into question small bilateral animal fossils; they thought some of the cellular structure interpreted by Chen et al. comes from diagenetic origin.4 The evidence from microfossils has been interpreted as the preserved gastrulae of cnidarians and bilateral metazoans, but could alternatively be interpreted as conventional algal cysts or egg cases modified by diagenetic processes.5Some researchers even considered that the cleavage globular fossils were not embryos but large sulfur bacteria.6 Anyway, no evidence could deny that metazoan and animal embryos existed in Doushantuo Formation. Most discussions are focused on whether some of the structure interpreted is of biological origin or not. The preservation of the fossils is very important for the interpretation of phosphatic fossils or even phylogenetic evolution. Encrustation and impregnation was considered the principle mode in phosphatization and calcification of animal soft tissue. Phosphatization appears to result from complex interactions between organic decay and mineral replication.7 Decay-resistant tissues such as cuticles and xylem are inclined to preserve easily. However, the protoplasm of the cells seems difficult to preserve. The crystallization of * To whom correspondence should be addressed. E-mail: [email protected] (H.C.), [email protected] (Z.L.). † State Key Laboratory of Bioelectronics, Southeast University. ‡ School of Biomedical and Engineering, Southeast University. § Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences. | Key Laboratory of Child Development and Learning Science, Southeast University.

phosphatic minerals always plays an important role in the fossilization process of Doushantuo fossils. Whole rock X-ray diffraction analysis shows that the phosphorites consist of apatite and dolomite with minor clay minerals.8 When Xiao et al.7 studied the preservation of fossils from Doushantuo Formation, they found that the crystal structure exists in both the fossil embryos and algae. Phosphatic crystals that occurred vertically on the envelope of the embryos were interpreted as coating, which helps to preserve the original structure. And coating was also found on both sides of the cell wall of the algae fossils. Phosphatic crystals found growing randomly inside the fossil cell lumens were interpreted as impregnation, most of which were the result of diagenesis. However, the molecular mechanism of the phosphatization is still unknown. When studying the structure of the fossils cells of algae and animal embryos by confocal laser scanning microscopy, we found that the fossil cells fluoresce when excited by laser light and have a steady fluorescent emission wavelength. Through three-dimensional reconstruction of the fossil cells, we got an image that could pass for one taken by a transmission light microscope. Quantitative fluorescence analysis was also done on the fossil algae.9 Meanwhile, Schopf et al. also used confocal laser scanning microscopy observing the Precambrian microorganisms preserved in stromatolitic cherts.10 Palaeontologists and geologists have found fluorescent fossils in other stratum by using a fluorescent microscope and a confocal laser scanning microscope. The algae layers were found with fluorescence in the Eocene Messel oil shale in Germany by means of fluorescent microscopy.11 Palynologists have used fossil pollen and spores for studying the geological time scale by employing fluorescent microscopy.12 Feistburkhardt and Pross13 have studied the Middle Jurassic dinoflagellate cyst marker species by using the confocal laser scanning microscope. Organic matter has been considered as the source of the fluorescence. In order to find out why Doushantuo fossil cells fluoresce, we observed the ultra structure of the minerals that replicated the ancient cells by confocal laser scanning microscopy and

10.1021/cg7008539 CCC: $40.75  2009 American Chemical Society Published on Web 12/08/2008

Apatite Crystal in Fossil Cells with Fluorescence

Crystal Growth & Design, Vol. 9, No. 2, 2009 677

Figure 1. Three-dimensional reconstruction of fossil algae cells under confocal laser scanning microscopy. (a, c) Part of algae of W. globosa; the cell wall was crystallized and it emitted fluorescence. (b) Possible cell island structure with fluorescence; we can see bigger fluorescent crystals as indicated by the arrows. Scale bar in (a) represents 50 µm for (a-c).

Figure 2. Three-dimensional reconstruction of putative fossil embryos scanned by confocal laser scanning microscopy. (a) A possible embryo in the 64-cell cleaving stage; (b, d) embryos without cleaving; (c) a possible 4-cell cleaving stage. Scale bar in (a) represents 200 µm for (a-d). The arrows in (a-c) indicate fluorescent crystal grains.

scanning electron microscopy. We found fluorescent apatite crystals in the fossil cells with different sizes and shapes. Using energy dispersive spectroscopy and powder X-ray diffraction, we confirmed that Ca5(PO4)3F crystals existed in the fossil cells, which could be the main reason causing the fossil cells to illuminate.

2. Materials and Methods 2.1. Sample Preparation for SEM Observation. The samples included in the present study were collected from the Weng‘an Biota, Doushantuo Formation in Guizhou, China. The age of the samples is 632-551 million years ago or 600 million years ago.3 The fossil rocks were cut into small pieces of 1-4 cm. They were put into 1% HCl for 5 s before removing the small rocks with an iron sieve. Then the rocks were washed with distilled water and immersed in dilute 9% acetic acid for 24-48 h on a 50 µm web, withdrawn from the acid, immersed sequentially in three baths of distilled water, and allowed to dry in air

conditioned atmosphere. The acid residue was collected and observed with a stereo light microscope, and the fossil embryos were picked out. 2.2. Sample Preparation for Confocal Laser Scanning Microscopy. The fossil rocks were cut into small square pieces and glued on the glass slide by epoxy resin. This rock section was polished until it became thin enough for observation in a light microscope in the transmission mode. Usually, the thickness of the fossils was about 50 µm. Using a LEICA TCS SP equipped with ArKr laser, which can emit light in 488, 476, and 568 nm, we observed the fossil specimens by 488 nm laser light. 2.3. SEM and Energy Dispersive Spectroscopy of Fossils. We chose about 50 globular fossils picked out from acid residue and scanned them under a leo-5100 scanning electron microscope. We also analyzed the elemental constituents of the selected areas by energy dispersive spectroscopy. 2.4. Powder X-ray Diffraction. We chose about 70 globular fossils from acid residue and crushed them in a smooth vessel until the fossil powder was fine enough to pass through a nylon-web with 5 µm in

678 Crystal Growth & Design, Vol. 9, No. 2, 2009

Chi et al.

Figure 3. Images of the fossil embryos taken by transmission light microscope. (a) The same fossil in Figure 2b; (b) the same fossil in Figure 2a.

Figure 4. Images of the fossil embryos scanned by scanning electron microscope. (a) Three-dimensional fossil embryo from acid residue represents a possible 64-cell cleaving stage; (b) enlargement of (a) as arrow 1 in (a) indicates; (c) enlargement of (a) as arrow 2 in (a) indicates; (d) enlargement of (a) as arrow 3 in (a) indicates. The arrows in (b-d) indicate the crystals growing on the surface of fossil cells. The scale bar in (a) represents 200 µm; the scale bar in (b) represents 2 µm for (b and c); the scale bar in (d) represents 5 µm. mesh diameter. The powder was collected and then put into the specimen chamber of a SHIMADZA DX-3A powder X-ray diffraction instrument. The data were analyzed with Pcpdfwin software.

3. Results and Discussion 3.1. The Fluorescent Fossil Cells under a Confocal Laser Scanning Microscope. We observed hundreds of algae fossil specimens and found almost all of them could fluoresce more or less when exited by laser light. Figure 1 shows representative fossil cells of algae. Through three-dimensional reconstruction of the fluorescent algae cells we can see the distinct profile of the cells indicated by Figure 1. Figure 1a

shows one part of a well-preserved Wengania globosa. 14 The fluorescent part was the cell wall, connecting with each other to form a web-like structure. The cell lumens inside the web have relatively weaker fluorescence. Figure 1c is also one part of W. globosa with bigger cells than that in Figure 1a. Figure 1b is the algae fossil with cell island structure, which was interpreted as a sexual structure of red algae.15 From Figure 1b we can see the big piece of fluorescent crystal as indicated by the arrows. We found not only the algae fossil cells but also the putative fossil embryos could fluoresce under our confocal laser scanning microscope.16 Figure 2 shows fluorescent fossil embryos in

Apatite Crystal in Fossil Cells with Fluorescence

Crystal Growth & Design, Vol. 9, No. 2, 2009 679

Figure 5. The fossil embryos with decorated envelope under scanning electron microscope. (a) An embryo without cleavage; (b) the enlargement of the envelope of (a); (c) an embryo in a possible 16-cell cleaving stage; (d) the enlargement of the envelope of (c); (e) the enlargement of (b) indicating the crystals growing on the envelope; (f) the enlargement of (d) indicating the crystals growing on the surface of envelope. The scale bar represents 200 µm in (a, c); 50 µm in (b, d); 1 µm in (e, f).

Figure 6. The fossil embryos scanned by scanning electron microscope. (a) Fossil embryo with decorated envelope; (b) enlargement of (a) in the arrow part of (a); the arrows in (b) show the crystals growing inside the embryo; (c) the perpendicular crystal growing inside the envelope. The scale bar represents 250 µm in (a), 100 µm in (b), 30 µm in (c).

Figure 7. The phosphatic crystals growing in a piece of residue from the acid. (a) The enlargement of the residue; (b) enlargement of (a); the crystals are hexagonal pillar traced in panel (b). The scale bar in (a) represents 30 µm; scale bar in (b) represents 5 µm.

different developmental stages. Figure 2b,d shows putative embryos in a noncleaving stage. Figure 2c is an embryo probably in a 4-cell cleaving stage. Figure 2a is an embryo in multicell cleaving stage. We can see the crystal particles as arrows

indicated in Figure 2a-c. However, the crystal surrounding the putative embryo in Figure 2d was uninterrupted. We could not see a distinct crystal particle inside it under the optical microscope. We consider that the intensity of the fluorescence

680 Crystal Growth & Design, Vol. 9, No. 2, 2009

Chi et al.

Figure 8. Energy dispersive spectroscopy of the crystals in Figure 4.

Figure 9. The powder XRD patterns of fossil embryos.

lies in the degree of crystallization of the fossil cells. The crystal particles could also be observed by transmission light microscopy indicated in Figure 3. We could see the crystal structure in the visible spectrum by transmission light from Figure 3a,b. From Figures 2 and 3 we know that the preservation of the embryos is different from that of the algae fossils shown in Figure 1. The cell wall of the algae fossil was connected to each other to form a whole part. However, the animal cell was mainly constructed by discontinuous crystal particles, which shows that the crystallization of the cells of algae is different from that of the animal cells. 3.2. The Crystal Structure Scanned by Scanning Electron Microscope. In order to observe the crystal structure in detail, we chose typical embryos from the acid residue and scanned them by scanning electron microscopy. Then we got the results of crystals growing on the surface of the embryos and inside the embryos. Figure 4 was a putative fossil embryo in the 64-cell cleaving stage, which was in the same developmental stage as the embryo in Figure 2a. From the enlargement of Figure 2a we could observe the crystals growing on the surface of the embryo. We found small club-shaped particles of 230 nm average width and 870 nm average length. Some particles are oriented from several centers indicated by Figure 4d, and other particles grow perpendicular on the surface indicated by arrows in Figure 4b,d. Xiao et al.7 has reported

perpendicular crystals bigger in size when they observed the envelope of the embryos. We also found such type of mineral particles when we observed the envelopes of embryos with decoration on the surface indicated by Figure 5, which have been reported as fossil embryos similar to embryos of Crustacean.1b We found that on the surface of the decorated envelope club-shaped particles also exist, which grow perpendicular on the surface. And from the cross section of the envelope we also found such perpendicular growing crystals, indicating by Figure 6c. Therefore, we considered the small crystals within 1 mm in size, which growing as a self-assembled pattern, play an important role in preserving the detailed structure of fossils. And also these crystals should be the source of the illuminating phenomenon of the fossil cells. In addition, we also found crystals growing inside the fossil embryo indicated in Figure 6a,b. From Figure 6b we could see the bigger crystals about 30 µm in diameter growing inside the cell lumens, which should be due to diagenetic mechanism during the preservation the fossil embryo. Such a phenomenon was described as an impregnation process in fossilization.7 In order to observe the growing of the phosphatic crystals we also scanned the nonfossil residue from the acid and found the crystals growing on a piece of residue, indicated by Figure 7. We traced the regular crystals and know that the crystals were

Apatite Crystal in Fossil Cells with Fluorescence

hexagonal pillars of several microns in length. Meanwhile, we studied the other crystals in fossil cells and found these crystals have also hexagonal potential. 3.3. Energy Dispersive Spectroscopy of the Crystals. After observing the crystal structure of the fossil cells we analyzed the elements composition of the crystals by using energy dispersive spectroscopy. We got the elemental composition of the crystals indicated by Figure 8. From Figure 8 we know the crystal was mainly composed by Ca, P, O, F elements, which was consistent with the results of the mineral composition of the phosphatic fossil bed reported.5a In order to confirm the crystal compound we used powder X-ray diffraction. 3.4. Powder X-ray Diffraction of the Crystals in the Fossil Cells. After the powder of the embryos was put into the sample chamber of the instrument, we got the diffraction results. The diffraction reference is as follows: V ) 40 Kv; I ) 30 mA; CPS ) 1 K. XRD powder patterns presented in Figure 9 indicated the diffraction information. We analyzed the diffraction data by Pcpdfwin software. And from three main peaks mode we found the crystal compound was Ca5(PO4)3F (the card number is #731727; #750915; and #770120, respectively). We performed qualitative analysis of the crystal structure by powder XRD to confirm fluorapatite crystal in the fossil cells. And from the cards in Pcpdfwin software we know the fluorapatite crystal was hexagonal, which was consistent with our observations about the crystals in the fossil cells.

4. Conclusion Our observations provide more evidence of encrustation (coating) and impregnation for the phosphatization mechanism. And we found that crystallization of fossil cells made the fossil algae and embryos illuminate. We confirmed by energy dispersive spectroscopy that the crystals in the fossil cells were mainly composed by Ca, O, P, and F elements. Furthermore, we found the crystals were fluorapatite by powder X-ray diffraction. Thus, we conclude that the micro crystals of Ca5(PO4)3F, rather than the organic matter, were the main fluorescent source of the fossil cells. Acknowledgment. We thank Prof. Chiawei Li, Miss Yuting Su, & Mr. Hunjen Wu in life science department of National Tsinghua University, Taiwan, for their kind help; we also thank Prof. Junyuan Chen in Nanjing Geology and Paleontology Institute. This work was supported by the National Science Foundation of China (Grant No. 60121101), jointly with the State Key Laboratory of Biomolecular Electronics, Southeast University and LPS, Nanjing Institute of Geology & Palaeontology, CAS.

Crystal Growth & Design, Vol. 9, No. 2, 2009 681 Supporting Information Available: We observed many globular fossils picked up from acid residues under the confocal fluorescent microscope and found that almost all the fossils fluoresce (Figure 1). We considered that it must be the materials on the surface of the globular fossils that emit the fluorescence. Then we found the phosphatic crystals on the surface of the fossils by SEM and even AFM (Figure 2). We observed dozens of the fluorescent globular fossils and found crystals exist on almost all the surface of fossils we selected. Thus, we think the crystals made the fossils fluoresce. Results of powder X-ray diffraction confirmed this idea. In order to ascertain whether organic carbon or other trace elements exist in the phosphatic crystals we performed element mapping by using the leo-5100 SEM. Figure 3 is a globular fossil in thin section and Figure 3b is the fluorescent image construction. Figure 3a is the image scanned by SEM. Figure 4 is the element mapping of 11 different trace elements including carbon. By comparing the distribution of fluorescence in Figure 3b and the distribution of element carbon we found they did not match very well. That means carbon could not be the main fluorescence source. So, the apatite crystals should more probably be the fluorescence source. This information is available free of charge via the Internet at http:// pubs.acs.org.

References (1) (a) Li, C. W.; Chen, J. Y.; Hua, T. Science 1998, 279, 879–882. (b) Xiao, S. H.; Zhang, Y.; Knoll, A. H. Nature 1998, 391, 553–558. (2) (a) Chen, J. Y.; Bottjer, D. J.; Oliveri, P.; Dornbos, S. Q.; Gao, F.; Ruffins, S.; Chi, H. M.; Li, C. W.; Davidson, E. H. Science 2004, 305, 218–222. (b) Yuan, X. L.; Xiao, S. H.; Taylor, T. N. Science 2005, 308, 1017–1020. (3) (a) Condon, D.; Zhu, M. Y.; Bowring, S.; Wang, W.; Yang, A. H.; Jin, Y. G. Science 2005, 308, 95–98. (b) Barfod, G. H.; Albare’de, F.; Knoll, A. H.; Xiao, S.; Te′Louk, P.; Frei, R.; Baker, J. Earth Planet. Sci. Lett. 2002, 201, 203–212. (4) (a) Bengtson, S.; Budd, G. Science 2004, 306, 1291a. (b) Chen, J. Y.; Oliveri, P.; Davidson, E. H.; Bottjer, D. J. Science 2004, 306, 1291b. (5) (a) Chen, J. Y.; Oliveri, P.; Li, C. W.; Zhou, G. Q.; Gao, F.; Hagadom, J. W.; Peterson, K. J.; Davidson, E. H. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 4457–4462. (b) Xiao, S. H.; Yuan, X. L.; Knoll, A. H. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13684–13689. (6) Bailey, J. V.; Joye, S. B.; Kalanetra, K. M.; Flood, B. E.; Corsetti, F. A. Nature 2007, 445, 198–201. (7) Xiao, S. H.; Knoll, A. H. Lethaia 1999, 32, 219–240. (8) Chen, D. F.; Dong, W. Q.; Zhu, B. Q.; Chen, X. P. Precambrian Res. 2004, 132, 123–132. (9) Chi, H. M.; Xiao, Z. D.; Fu, D. G.; Lu, Z. H. Microsc. Res. Tech. 2006, 69, 253–259. (10) Schopf, J. W.; Tripathi, A. B.; Kudryavtsev, A. B. Astrobiology 2006, 6, 1–16. (11) Nix, T.; Feist-burkhardt, S. Geol. Mag. 2003, 140, 469–478. (12) Yeloff, D.; Hunt, C. ReV. Palaeobot. Palynol. 2005, 133, 203–219. (13) Feist-burkhardt, S.; Pross, J. Bull. Cent. Rech. Elf Explor. Prod. 1998, 22, 103–45. (14) Zhang, Y. Lethaia 1989, 22, 113–132. (15) Zhang, Y.; Yuan, X. L. Sci. China (Series C) 1996, 39, 27–36. (16) Chi, H. M.; Feng, M.; Xiao, Z. D.; Lu, Z. H. Microsc. Res. Tech. 2008, 71 (4), 260–266.

CG7008539