Photonic techniques are helping researchers dig deeper than ever into the past, unearthing new findings about some of the oldest — as well as some of the largest — skeletonized organisms known in the geological record. Raman spectroscopy and confocal scanning laser microscopy have revealed details about fossils from the early Cambrian period, offering hints as to the evolutionary history of animals. At the same time, mass spectrometry has contributed to analyses of soft tissue from mastodon and Tyrannosaurus rex, helping to obtain genome sequences that could provide information on early evolutionary links.Using mass spectrometry, researchers have shown that they can obtain genome sequences from collagen found in the fossilized bone of a mastodon and a Tyrannosaurus rex. The latter yielded the oldest protein sequenced; previously, investigators had believed that the fossilization process eventually destroys all original organic components. Courtesy of Zina Deretsky, National Science Foundation.Finding some backboneThe 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. Understanding the earliest radiation of the Cambrian population rise comes primarily from study of disconnected microscopic skeletal components — or “small shelly fossils” — found in the assemblage. These fossils typically lack identifiable soft tissue, however, and as a result offer only 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 (left) and a Raman image (middle), in which the varying intensities correspond to the concentrations of the molecular structures detected. Confocal laser scanning microscopy images revealed the anatomical structure of the embryo (right). The scale bar is 25 μm. AC = aboral canal; AO = apical (aboral) organ; EM = egg membrane; MC = meridional canal; CT = ctenes (“comb rows”). Reprinted with permission of PNAS.Several collections in southern China also contain metazoan eggs, the study of which has contributed significantly to our knowledge of evolutionary developments in the Cambrian period. An excellently preserved late-stage embryo of a ctenophore was recently found in southwestern Shaanxi. In the April 10 issue of PNAS, a group with Nanjing University in China and with the University of California and the University of Southern California, both in Los Angeles, reported characterization of the embryo using Raman spectrometry and confocal scanning laser microscopy. The study provided information on the early evolution of ctenophores and, thus, insight into one of the oldest complex organisms in the geological record.Researcher David J. Bottjer has been working with investigator Jun-Yuan Chen to study the Cambrian fossils. “We also had been aware for some time of [J. William] Schopf’s ongoing work using Raman and confocal techniques,” Bottjer said. “Schopf 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.”Schopf noted that the techniques, both of which are new to paleobiology, are complementary. “Both provide data that, taken together, solve two long-standing problems in the field: (1) an inability to document accurately the three-dimensional morphology of permineralized fossils at micron-scale spatial resolution; and (2) the lack of a means to analyze in situ and, at such resolution, the chemistry of the coaly matter (kerogen) that comprises the structurally preserved anatomy of such fossils.”He added that both methods measure signals derived from properties of the kerogenous materials in question: for confocal, laser-induced fluorescence derived primarily from the interlinked polycystic aromatic hydrocarbons that predominate in kerogen; for Raman, it was laser-induced vibrational transitions in the bonds of these polycystic aromatic hydrocarbons and their associated functional groups. Furthermore, both are nonintrusive and nondestructive. “Together,” he said, “they provide data unavailable by any other means.”The ctenophore embryo described in the PNAS paper was preserved in a 10-cm-thick bed of phosphatic limestone; it is the earliest known example of the phylum. 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 thin section. Discovery and study of ancient fossils such as this can yield information about the early ontogenetic development of metazoans and can offer a glimpse into the early evolutionary history of animals.First, the researchers used a triple-stage laser Raman system made by Jobin Yvon Horiba 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 about 1.5 μm and vertical resolution of about 2 to 3 μm; a 100×, 0.8-NA objective achieved horizontal resolution of about 0.7 μm and vertical resolution of about 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 investigators performed two-dimensional imaging of the specimen by covering the fossilized embryo 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 of Center Valley, Pa., and then analyzing it. This demonstrated the means by which the specimens were preserved: Embryos preserved in some of the eggs were permineralized in a combination of phosphate and kerogen — carbonaceous remnants of the original organic components of the embryos — aided by secondarily emplaced calcite.They revealed the anatomical structure of the ctenophore with confocal laser scanning microscopy. They obtained three-dimensional confocal fluorescence images using an Olympus Fluoview 300 confocal laser scanning biological microscope system with a BX51 microscope and a 488-nm argon-ion laser from Melles Griot of Carlsbad, Calif.The researchers used the Raman system because it could do everything (at least to date) that they hoped it could do, Schopf noted. Importantly, the system enabled two-dimensional imaging at micron-scale resolution. “This was new to paleobiology when we introduced it in 2002,” he said. Moreover, using the 2-D images, the investigators figured out how to acquire 3-D Raman images.Schopf noted that his lab purchased the Olympus confocal system because trials had convinced him that it would perform the specific tasks he had in mind, because it is easily available in Los Angeles, and because its optics are compatible/interchangeable with those of the previously acquired Raman system. Other systems likely would have worked, but because Schopf was not working with living organisms, he did not need all that is required for imaging living cells.The findings of the study are significant. The researchers were able to understand the composition of the early animal embryo fossils at a greater resolution than with previous studies and were able to document that the fossil in question was indeed a ctenophore embryo.They continue to apply the techniques to fossils of plants, fungi and algae as well as of animals. Also, Schopf said, “in the next six months, we will be investigating new materials from India and Russia — in collaboration with scientists visiting my lab from those countries — and attempting to even better define the applicability of these techniques to understanding the history and evolution of life.”Checking out the lizard kingStudying fossils with photonic techniques has helped to glean understandings of some early animals, from the oldest to some of the largest. In a recent report, researchers described using mass spectrometry to obtain genome sequences from the fossilized bones of mastodon and T. rex.Acquiring genome sequences from a variety of taxa has contributed significantly to understanding evolution and adaptation. As researchers dig deeper into the past, though, studies of molecular evolution are sometimes hampered by the challenges inherent to obtaining DNA or RNA from ancient, extinct taxa. Sequence data from fossilized bone 1 million years old or older has proved especially elusive — because of the limits of detection of most analytical methods and commercial software’s reliance on identical matches between peptide fragmentation patterns and existing peptide/protein sequences in available databases, as well as the potential for degradation over such long periods.In the April 13 issue of Science, researchers from Beth Israel Deaconess Medical Center and Harvard Medical School, both in Boston, from North Carolina State University and North Carolina Museum of Natural Sciences, both in Raleigh, and from Montana State University in Bozeman reported two studies in which they addressed these limitations in fossilized bone from a 160,000- to 600,000-year-old mastodon and a 68 million year-old T. rex — the latter yielding the oldest protein sequenced.The first question they addressed was whether it is even possible to acquire protein sequence data from fossils 1 million years old or older. Researchers have long believed that almost all original organic components are destroyed during the process of fossilization. In one of the Science papers, “Analyses of Soft Tissue from Tyrannosaurus rex Suggest the Presence of Protein,” the investigators showed that collagen I, the main organic component of bone, had been preserved in the tissue — albeit in very low concentrations. They identified the collagen using several methods, including immunohistochemistry as well as transmission electron and atomic force microscopy.Still, they wanted to know more than whether protein was present in fossils that are 1 million years old or older. “We had accumulated reams of ‘circumstantial evidence’ that proteins persisted [for so long],” said researcher Mary H. Schweitzer, “but the nail in the coffin, the ‘holy grail’ of molecular paleontology remains sequence data.” The investigators knew that they had tremendously low amounts of protein and that there was a significant amount of contamination. “I knew that we would need technology of the highest sensitivity and accuracy, and mass spectrometry technology has achieved that level.”In the other Science paper, “Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry,” the researchers described a two-step proteomics approach with which to overcome the obstacles associated with acquiring sequence data from fossils that are 1 million years old or older. First, they found tryptic peptide fragments from the fossilized bone that were identical matches with sequences from different, extant taxa deriving from a common ancestor (known as orthologs), serving to identify the protein of interest. This procedure already is used widely with proteins from taxa that share genomic information with other taxa.Next, by comparing amino acid sequences of orthologs from a number of related extant species, the scientists compiled a protein sequence database of probable drifts in amino acids in other tryptic proteins. The database thus provided a workable number of theoretical protein sequences. These yielded predicted peptide fragmentation patterns that the investigators could compare with peptide fragments from fossilized bone with no matches in public sequence databases.They acquired the mass spectra using a linear ion trap mass spectrometer made by Thermo Electron Corp. of San Jose, Calif., operating in positive ion mode for data-dependent acquisitions. The spectra were then searched against a publicly available National Institutes of Health database; for the mastodon bone fragments, the researchers used an additional database, from the European Molecular Biology Laboratory and Sanger Institute in Cambridge, UK.“The linear ion trap is more sensitive than most mass spectrometers, for several reasons,” said researcher John M. Asara. “In general, ion traps have the ability to store ions over time, which can build up signal intensity.” He added that the Thermo Electron instrument’s dual ejection and two detectors make the ion transmission and detection more efficient, contributing to maximum sensitivity — though these advantages come at the expense of mass accuracy and resolution. “TOF [time of flight]-based instruments have higher resolution and mass accuracy, but they are slightly less sensitive, since the ions are detected as they pass through the instrument without the ion storage capability.”Purifying peptidesThe researchers faced a variety of challenges during the study. “As far as T. rex goes,” Asara noted, “the biggest challenge was purifying the peptide component from 99.99 percent of a brown gritty contaminant. Based on tricks I learned with microchromatography in a 2002 mammoth study, we were able to use cation exchange to separate the peptides from the contamination. This process went through many extractions over a year and a half.” He attributes the success of the study largely to the combination of the purification techniques and the sensitive mass spectrometer as well as to the bioinformatics method they developed to uncover novel sequences unique to the organisms of interest.The researchers developed a proteomics approach that enabled them to match sequences from the fossilized bone of a T. rex (MOR 1125, or Museum of the Rockies specimen 1125) with those from different, extant species. This process showed that chickens, newts and frogs are the dinosaur’s closest relatives.In the end, the scientists showed that the two-step proteomics approach can provide sequence information for both mastodon and T. rex. “That sequenceable organic material exists as far out as 68 million years is astonishing,” Asara said. For the mastodon bone fragments, the scientists matched the mass spectra to collagen sequences from extant mammals in the protein database. For the T. rex, they sequenced seven peptides of collagen, and in matching the sequences to the database, showed that chickens, newts and frogs are the dinosaur’s closest relatives, confirming hypotheses about the chicken sequence homology.The researchers plan to continue studying the fossils and hope to unearth more extensive protein sequences to establish better evolutionary relationships. Schweitzer noted that further fieldwork is another important goal. “Need more dinosaur bones!” she exclaimed.