The Meishucun assemblage of fossils in southwestern Shaanxi, China, documents the beginnings of the Cambrian explosion of animals roughly 540 million years ago — a period in which the diversity of skeletonized metazoans (multicellular animals that are more advanced than sponges) rose markedly and rapidly. A current understanding of the earliest radiation of the Cambrian development largely comes from the study of disconnected microscopic skeletal components — or “small shelly fossils” — that are found in the assemblage. These fossils typically lack identifiable soft tissue, however, and offer only a limited understanding of the earliest stages of metazoan evolution.Using Raman spectrometry and confocal laser scanning microscopy, researchers have characterized fossilized ctenophore embryos roughly 540 million years old, providing insight into one of the oldest complex organisms in the geological record. Shown here are an optical image (a), confocal laser scanning microscopy images (b, c, g), Raman spectra (d) and Raman images (e, g, h, i, j) of an embryo. AC = aboral canal. The numbers 1 through 8 indicate the comb rows, or rows of cilia, attached to the embryo.Several assemblages in southern China also contain metazoan eggs, the study of which has contributed in significant ways to understandings of the Cambrian explosion. Investigators recently found an excellently preserved late-stage embryo of a ctenophore, or “comb jelly.”David J. Bottjer of the University of Southern California in Los Angeles has been studying the Cambrian fossils with Jun-Yuan Chen of Nanjing University in China. “We also had been aware for some time of [J. William] Schopf’s ongoing work using Raman and confocal techniques,” Bottjer said. “Schopf [of the University of California, Los Angeles] has used these techniques to study Precambrian microfossils — primarily ones of microbial origin. We all agreed that using Raman and confocal on some of Chen’s fossils with excellent preservation would be a good research direction to take.” The techniques’ potential for compositional determination and three-dimensional imaging made them particularly appealing to the investigators.Ctenophore embryoThe earliest known example of the phylum, the ctenophore embryo was preserved in a 10-cm-thick bed of limestone. The egg with the embryo inside it is spheroidal and about 190 μm in diameter; the embryo itself is about 150 μm in diameter and was prepared in a 50-μm-thick section.First, the researchers used a triple-stage laser Raman system made by Horiba Jobin Yvon of Edison, N.J., to explore the processes that led to preservation of the eggs. A 50×, 0.5-NA objective offered horizontal resolution of ~1.5 μm and vertical resolution of ~2 to 3 μm; a 100×, 0.8-NA objective provided a horizontal resolution of ~0.7 μm and vertical resolution of ~1.0 to 1.5 μm. An argon-ion laser made by Coherent Inc. of Santa Clara, Calif., provided excitation at 457.9 nm.The scientists performed two-dimensional imaging of the specimen by covering it with a thin layer of fluorescence-free microscopy immersion oil, centering the fossil in the path of the beam as it was transmitted through a microscope made by Olympus America Inc. of Center Valley, Pa., and then analyzing the specimen. They found that embryos preserved in some of the eggs were permineralized with a combination of phosphate and kerogen — carbonaceous remnants of the original organic components of the embryos — aided by secondarily emplaced calcite.They then revealed the anatomical structure of the ctenophore with confocal laser scanning microscopy, obtaining three-dimensional confocal fluorescence images using an Olympus microscope system and a 488-nm argon-ion laser made by Melles Griot of Carlsbad, Calif.The findings were significant. “We were able to understand the composition of these early animal embryo fossils at a greater resolution than has been understood with previous studies and to document that this fossil is indeed a ctenophore embryo,” Bottjer said.The investigators will continue to look for fossil specimens. Applying the techniques to other fossils will provide further insight into the early evolution of animals, he added.PNAS, April 10, 2007, pp. 6289-6292.