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Article
Published: 2021-06-29

Application of confocal laser scanning microscopy to the study of amber bioinclusions

State Key Laboratory of Palaeobiology and Stratigraphy, Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China; University of Chinese Academy of Science, Beijing 100049, China
State Key Laboratory of Palaeobiology and Stratigraphy, Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China
State Key Laboratory of Palaeobiology and Stratigraphy, Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China
State Key Laboratory of Palaeobiology and Stratigraphy, Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China
State Key Laboratory of Palaeobiology and Stratigraphy, Center for Excellence in Life and Paleoenvironment, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing 210008, China
General CLSM imaging system fluorescence micro-CT fossil arthropod inclusions

Abstract

Confocal laser scanning microscopy is an essential analytical tool in biological, biomedical, and material sciences, integrating microscope manufacturing technology, optical-electronic technology, and computer technology. In the last decade, confocal laser scanning microscopy has been successfully applied to the study of amber bioinclusions. Enhanced signal to noise ratios, resolution power, capability of optical sectioning, three-dimensional reconstruction, and better performance when imaging thicker samples provide a great deal of valuable and detailed morphological information about amber fossils. We briefly discuss the practical applications of CLSM in amber studies and compare it with other imaging methods commonly used in the field, including bright-field microscopy, wide-field fluorescence microscopy, and micro-computed tomography. A general procedure for imaging amber inclusions with CLSM is provided, with a focus on pretreatments and image processing.

References

  1. Ascaso, C., Wierzchos, J., Corral, J.C., Lopez, C. & Alonso, J. (2003) New application of light and electron microscopic techniques for the study of microbial inclusions in amber. Journal of Paleontology, 77, 986–996. https://doi.org/10.1666/0022-3360(2003)077<1182:NAOLAE>2.0.CO;2
  2. Ascaso, C., Wierzchos, J., Speranza, M., Gutiérrez, J.C., González, A.M., de los Ríos, A. & Alonso, J. (2005) Fossil protists and fungi in amber and rock substrates. Micropaleontology, 51, 59–72. https://doi.org/10.2113/51.1.59
  3. Arillo, A., Subías, L.S. & Sánchez-García, A. (2016) New species of fossil oribatid mites (Acariformes, Oribatida), from the Lower Cretaceous amber of Spain. Cretaceous Research, 63, 68–76. https://doi.org/10.1016/j.cretres.2016.02.009
  4. Azar, D., Perrichot, V., Neraudeau, D. & Nel, A. (2003) New psychodid flies from the Cretaceous ambers of Lebanon and France, with a discussion about Eophlebotomus connectens Cockerell, 1920 (Diptera, Psychodidae). Annals of the Entomological Society of America, 96, 117–127. https://doi.org/10.1603/0013-8746(2003)096[0117:NPFTCA]2.0.CO;2
  5. Bao, T., Wang, B., Li, J.G. & Dilcher, D. (2019) Pollination of Cretaceous flowers. Proceedings of the National Academy of Sciences, USA, 116, 24707–24711. https://doi.org/10.1073/pnas.1916186116
  6. Böker, C. & Brocksch, D. (2002) Fascinating amber. Carl Zeiss: Innovation, 12, 20–23.
  7. Borlinghaus, R.T. (2017) The white confocal: microscopic optical sectioning in all colours. Springer International Publishing, Cham, 115 pp. https://doi.org/10.1007/978-3-319-55562-1
  8. Bonato, L., Edgecombe, G.D. & Minelli, A. (2014) Geophilomorph centipedes from the Cretaceous amber of Burma. Palaeontology, 57, 97–110. https://doi.org/10.1111/pala.12051
  9. Cai, C.Y. & Huang, D.Y. (2014) The oldest micropepline beetle from Cretaceous Burmese amber and its phylogenetic implications (Coleoptera: Staphylinidae). Naturwissenschaften, 101, 813–817. https://doi.org/10.1007/s00114-014-1221-z
  10. Cai, C.Y., Clarke, D.J., Yin, Z.W, Fu, Y.Z. & Huang, D.Y. (2019) A specialized prey-capture apparatus in mid-Cretaceous rove beetles. Current Biology, 29, R105–R119. https://doi.org/10.1016/j.cub.2019.01.002
  11. Cai, C.Y., Thayer, M.K., Newton, A.F., Yin, Z.W. & Huang, D.Y. (2018) A new genus of dasycerine rove beetles from Upper Cretaceous Burmese amber and its phylogenetic implications (Coleoptera, Staphylinidae). Cretaceous Research, 84, 431–436. https://doi.org/10.1016/j.cretres.2017.12.004
  12. Chen, D.J., Yang, J. & Shao, Y.W. (2019) Amber insects. Chemical Industry Press, Beijing, pp. 1–274 [in Chinese].
  13. Christiansen, K. & Nascimbene, P. (2006) Collembola (Arthropoda, Hexapoda) from the mid Cretaceous of Myanmar (Burma). Cretaceous Research, 27, 318–363. https://doi.org/10.1016/j.cretres.2005.07.003
  14. Claxton, N.S., Fellers, T.J. & Davidson, M.W. (2006) Laser scanning confocal microscopy. In: Webster, J.G. (Ed.), Encyclopedia of medical devices and instrumentation. New Jersey, John Wiley & Sons. pp. 449–477. https://doi.org/10.1002/0471732877.emd291
  15. Clark, N.D.L. & Daly, C. (2010) Using confocal laser scanning microscopy to image trichome inclusions in amber. Journal of Paleontological Techniques, 8, 1–7.
  16. Cox, G. & Sheppard, C.J. (2004) Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research and Technique, 63, 18–22. https://doi.org/10.1002/jemt.10423
  17. Cruickshank, R.D. & Ko, K. (2003) Geology of an amber locality in the Hukawng Valley, northern Myanmar. Journal of Asian Earth Sciences, 21, 441–455. https://doi.org/10.1016/S1367-9120(02)00044-5
  18. Fu, Y.Z., Azar, D. & Huang, D.Y. (2020) A new species of the extinct family Minlagerrontidae (Insecta: Hemiptera: Cicadomorpha) from mid-Cretaceous Burmese amber. Cretaceous Research, 107, 104270. https://doi.org/10.1016/j.cretres.2019.104270
  19. Fu, Y.Z., Szwedo, J. & Huang, D.Y. (2021) A new sinoalid froghopper in mid-Cretaceous amber from northern Myanmar (Hemiptera, Cicadomorpha, Sinoalidae). Cretaceous Research, 125, 104841. https://doi.org/10.1016/j.cretres.2021.104841
  20. González, S. & Halpern, A. (2007) Laser-scanning confocal microscopy. In: Soyer, H.P., Argenziano, G., Hofmann-Wellenhof , R. & Johr, R.H. (Eds), Colour atlas of melanocytic lesions of the skin. Springer, Berlin, Heidelberg, pp. 39–46. https://doi.org/10.1007/978-3-540-35106-1_5
  21. Compton, S.G., Ball, A.D., Collinson, M.E., Hayes, P., Rasnitsyn, A.P. & Ross, A.J. (2010) Ancient fig wasps indicate at least 37 Myr of stasis in their mutualism with fig trees. Biology Letters, 6, 838–842. https://doi.org/10.1098/rsbl.2010.0389
  22. Guo, M.X., Yang, H.D., Li, G., Tong, Y.-J., Li, S., Lu, Y.Y., Shi, A.M., Wang, B., Zhang, W.W. & Bai, M. (2016) Morphological identifiability of Burmese amber inclusions under X-rays. Acta Entomologica Sinica, 59, 1013–1020 [In Chinese]. https://doi.org/10.16380/j.kcxb.2016.09.012
  23. Halbhuber, K.J. & Konig, K. (2003) Modern laser scanning microscopy in biology, biotechnology and medicine. Annals of Anatomy, 185, 1–20. https://doi.org/10.1016/S0940-9602(03)80002-X
  24. Heethoff, M., Helfen, L. & Norton, R.A. (2009) Description of Neoliodes dominicus n. sp. (Acari, Oribatida) from Dominican amber, aided by synchrotron X-ray microtomography. Journal of Paleontology, 83, 153–159. https://doi.org/10.1666/08-101R1.1
  25. Hein, H.J., Czurratis, P., Schroth, D. & Bernstein, A. (1995) A comparative study of the application of scanning acoustic microscopy and confocal laser scanning microscopy to the structural assessment of human bones. Annals of Anatomy, 177, 427–430. https://doi.org/10.1016/S0940-9602(11)80149-4
  26. Hovis, D.B. & Heuer, A.H. (2010) The use of laser scanning confocal microscopy (LSCM) in materials science. Journal of Microscopy, 240, 173–180. https://doi.org/10.1111/j.1365-2818.2010.03399.x
  27. Kirejtshuk, A.G., Chetverikov, P.E. & Azar, D. (2015) Libanopsinae, new subfamily of the family Sphindidae (Coleoptera, Cucujoidea) from Lower Cretaceous Lebanese amber, with remarks on using confocal microscopy for the study of amber inclusions. Cretaceous Research, 52, 461–479. https://doi.org/10.1016/j.cretres.2014.02.008
  28. Kundrata, R., Bukejs, A., Prosvirov, A.S. & Hoffmannova, J. (2020) X-ray micro-computed tomography reveals a unique morphology in a new click-beetle (Coleoptera, Elateridae) from the Eocene Baltic amber. Scientific Reports, 10, 20158. https://doi.org/10.1038/s41598-020-76908-3
  29. Kypke, J.L. & Solodovnikov, A. (2020) Every cloud has a silver lining: X-ray micro-CT reveals Orsunius rove beetle in Rovno amber from a specimen inaccessible to light microscopy. Historical Biology, 32, 940–950. https://doi.org/10.1080/08912963.2018.1558222
  30. Lak, M., Néraudeau, D., Nel, A., Cloetens, P., Perrichot, V. & Tafforeau, P. (2008) Phase contrast X-ray synchrotron imaging: opening access to fossil inclusions in opaque amber. Microscopy and Microanalysis, 14, 251–259. https://doi.org/10.1017/S1431927608080264
  31. Li, Y.D., Yamamoto, S., Huang, D.Y. & Cai, C.Y. (2020a) Confocal and micro-CT data of Miniomma chenkuni, holotype, NIGP173375. Zenodo. https://doi.org/10.5281/zenodo.3994920
  32. Li, Y.D., Yamamoto, S., Huang, D.Y. & Cai, C.Y. (2020b) A miniaturized ommatid beetle in mid-Cretaceous Burmese amber (Coleoptera: Archostemata: Ommatidae). Papéis Avulsos de Zoologia, 60, e20206063. https://doi.org/10.11606/1807-0205/2020.60.63
  33. Li, Y.D., Tihelka, E., Leschen, R.A.B., Yu, Y., Ślipiński, A., Pang, H., Huang, D.Y., Kolibáč, J. & Cai, C.Y. (2021a) Confocal and micro-CT data of Microtrogossita qizhihaoi, holotype, NIGP173910. Zenodo.
  34. Li, Y.D., Yamamoto, S., Huang, D.Y. & Cai, C.Y. (2021b) Confocal and micro-CT data of Paraodontomma leptocristatum, holotype, NIGP174676. Zenodo. https://doi.org/10.5281/zenodo.4737033
  35. Li, Y.D., Yamamoto, S., Huang, D.Y. & Cai, C.Y. (2021c) New species of Paraodontomma from mid-Cretaceous Burmese amber with muscle tissue preservation (Coleoptera: Archostemata: Ommatidae). Papéis Avulsos de Zoologia, 61, e20216153. https://doi.org/10.11606/1807-0205/2021.61.53
  36. Li, Y.D., Huang, D.Y. & Cai, C.Y. (2021d) Confocal data of Pseudomataeopsephus burmensis, holotype, NIGP173913. Zenodo.
  37. Liu, Y.M., Hakim, M. & Huang, D.Y. (2020) First stratiomyomorphan larvae in the mid-Cretaceous amber from Myanmar (Diptera: Brachycera). Cretaceous Research, 106, 104256. https://doi.org/10.1016/j.cretres.2019.104265
  38. Martišek, D. (2017) 3D reconstruction of the surface using a standard camera. Mathematical Problems in Engineering, 2017, 4642397. https://doi.org/10.1155/2017/4642397
  39. Paddock, S.W. (2000) Principles and practices of laser scanning confocal microscopy. Molecular Biotechnology, 16, 127–149. https://doi.org/10.1385/MB:16:2:127
  40. Perreau, M. & Tafforeau, P. (2011) Virtual dissection using phase-contrast X-ray synchrotron microtomography: reducing the gap between fossils and extant species. Systematic Entomology, 36, 573–580. https://doi.org/10.1111/j.1365-3113.2011.00573.x
  41. Peyrot, D., Barrón, E., Pereda-Suberbiola, X. & Company, J. (2020) Vegetational composition of the Upper Cretaceous vertebrate site of Chera (Valencia, Spain) and its significance in mosaic vegetation from southwestern Europe. Cretaceous Research, 106, 104254. https://doi.org/10.1016/j.cretres.2019.104254
  42. Prasad, V., Farooqui, A., Murthy, S., Sarate, O.S. & Bajpai, S. (2018) Palynological assemblage from the Deccan Volcanic Province, central India: insights into early history of angiosperms and the terminal Cretaceous paleogeography of peninsular India. Cretaceous Research, 86, 186–198. https://doi.org/10.1016/j.cretres.2018.03.004
  43. Robin, N., Béthoux, O., Sidorchuk, E., Cui, Y., Li, Y., Germain, D., King, A., Berenguer, F. & Ren, D. (2016) A Carboniferous mite on an insect reveals the antiquity of an inconspicuous interaction. Current Biology, 26, 1376–1382. https://doi.org/10.1016/j.cub.2016.03.068
  44. Ross, A.J. (2019) Burmese (Myanmar) amber checklist and bibliography 2018. Palaeoentomology, 2 (1), 22–84. https://doi.org/10.11646/palaeoentomology.2.1.5
  45. Ross, A.J. (2021) Supplement to the Burmese (Myanmar) amber checklist and bibliography, 2020. Palaeoentomology, 4 (1), 57–76. https://doi.org/10.11646/palaeoentomology.4.1.11
  46. Shihavuddin, A., Basu, S., Rexhepaj, E., Delestro, F., Menezes, N., Sigoillot, S.M., Nery, E.D., Selimi, F, Spassky, N. & Genovesio, A. (2017) Smooth 2D manifold extraction from 3D image stack. Nature Communications, 8, 15554. https://doi.org/10.1038/ncomms15554
  47. Sidorchuk, E.A. (2013) New technique for preparation of small-sized amber samples with application to mites. In: Azar, D., Engel, M.S., Jarzembowski, E., Krogmann, L., Nel, A. & Santiago-Blay, J.A. (Eds., .Insect Evolution in an amberiferous and stone alphabet. Proceedings of the 6th International Congress on Fossil Insects, Arthropods and Amber. Brill, Leiden-Boston. pp. 189–201. https://doi.org/10.1163/9789004210714_014
  48. Sidorchuk, E.A. & Vorontsov, D.D (2018) Preparation of small-sized 3D amber samples: state of the technique. Palaeoentomology, 1 (1), 80–90. https://doi.org/10.11646/palaeoentomology.1.1.10
  49. Speranza, M., Wierzchos, J., Alonso, J., Bettucci, L., Martín-González, A. & Ascaso, C. (2010) Traditional and new microscopy techniques applied to the study of microscopic fungi included in amber. In: Méndez-Vilas, A. & Díaz, J. (Eds) Microscopy: science, technology, applications and education. Badajoz, Spain, pp. 1135–1145.
  50. Su, Y.T., Cai, C.Y. & Huang, D.Y. (2019) Revision of Phryssonotus burmiticus (Diplopoda, Polyxenida, Synxenidae) in mid-Cretaceous amber from Myanmar. Cretaceous Research, 93, 216–224. https://doi.org/10.1016/j.cretres.2018.09.002
  51. Su, Y.T., Cai, C.Y. & Huang, D.Y. (2020) Two new species of the bristle millipede genus Pauropsxenus (diplopoda, Polyxenidae) in mid-Cretaceous Burmese amber. Cretaceous Research, 111, 104427. https://doi.org/10.1016/j.cretres.2020.104427
  52. Tihelka, E., Huang, D.Y. & Cai, C.C. (2020) New data on Ommatidae (Coleoptera) from mid-Cretaceous Burmese amber. Cretaceous Research, 106, 104253. https://doi.org/10.1016/j.cretres.2019.104253
  53. Tihelka, E., Li, L.Q., Fu, Y.Z., Su, Y.T., Huang, D.Y. & Cai, C.Y. (2021) Angiosperm pollinivory in a Cretaceous beetle. Nature Plants, 7, 445–451. https://doi.org/10.1038/s41477-021-00893-2
  54. Van de Kamp, T., Dos Santos Rolo, T., Baumbach, T. & Krogmann, L. (2014) Scanning the past-synchrotron X-ray microtomography of fossil wasps in amber. Entomologie heute, 26, 151–160.
  55. Xiao, Y.M., Fu, D.L. & Li, A.S. (1999) Laser scanning confocal microscope (LSCM) and its application in biology. Acta Laser Biology Sinica, 8, 305–311 [in Chinese]. https://doi.org/10.1007/BF02946523
  56. Yin, Z.W., Chandler, D.S. & Cai, C.Y. (2019) Priscaplectus gen. nov. and two new species in mid-Cretaceous amber from Myanmar (Coleoptera: Staphylinidae: Pselaphinae). Cretaceous Research, 103, 104174. https://doi.org/10.1016/j.cretres.2019.07.004
  57. Yin, Z.W., Lü, L., Yamamoto, S, Thayer, M.K., Newton, A.F. & Cai, C.Y. (2021) Dasycerinae rove beetles: Cretaceous diversification, phylogeny and historical biogeography (Coleoptera: Staphylinidae: Dasycerinae). Cladistics, 37, 185–210. https://doi.org/10.1111/cla.12430
  58. Zhang, Q.Q., Zhang, J.F. & Wang, B. (2017) First record of the subfamily Archinemestriinae in the family Nemestrinidae (Diptera: Brachycera) from Upper Cretaceous Burmese amber. Cretaceous Research, 75, 141–145. https://doi.org/10.1016/j.cretres.2017.03.005
  59. Zhao, X.D, Zhang, Q.Q., Jarzembowski, E.A., Chen, L. & Wang, B. (2016) A new earwingfly from mid-Cretaceous Burmese amber (Mecoptera: Meropeidae). Cretaceous Research, 66, 136–140. https://doi.org/10.1016/j.cretres.2016.06.008