Abstract Fluorescence using ultraviolet (UV) light has seen increased use as a tool in paleontology over the last decade. Laser-stimulated fluorescence (LSF) is a next generation technique that is emerging as a way to fluoresce paleontological specimens that remain dark under typical UV. A laser’s ability to concentrate very high flux rates both at the macroscopic and microscopic levels results in specimens fluorescing in ways a standard UV bulb cannot induce. Presented here are five paleontological case histories that illustrate the technique across a broad range of specimens and scales. Novel uses such as back-lighting opaque specimens to reveal detail and detection of specimens completely obscured by matrix are highlighted in these examples. The recent cost reductions in medium-power short wavelength lasers and use of standard photographic filters has now made this technique widely accessible to researchers. This technology has the potential to automate multiple aspects of paleontology, including preparation and sorting of microfossils. This represents a highly cost-effective way to address paleontology's preparatory bottleneck.

Citation: Kaye TG, Falk AR, Pittman M, Sereno PC, Martin LD, Burnham DA, et al. (2015) Laser-Stimulated Fluorescence in Paleontology. PLoS ONE 10(5): e0125923. https://doi.org/10.1371/journal.pone.0125923 Academic Editor: Christof Markus Aegerter, University of Zurich, SWITZERLAND Received: January 8, 2015; Accepted: March 26, 2015; Published: May 27, 2015 Copyright: © 2015 Kaye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Dr. Stephen S. F. Hui Trust Fund (201403173007), Faculty of Science and Department of Earth Sciences of the University of Hong Kong, the National Natural Science Foundation of China (41120124002), the 973 (National Basic Research) program (2012CB821900) and a Panorama grant supplied by the University of Kansas Natural History Museum and Biodiversity Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Highlighting and identifying fossilized structures can be difficult whether it is bone, soft tissue such as skin, muscle and internal organs, or integument such as scales and feathers. Historically, multiple methods have been used to highlight structures for photography, including cross-lighting, polarized light [1], camera filters, and ultraviolet (UV) light [2–6]. Cross-lighting can highlight structures that are difficult to see in direct light. Polarized light can help to enhance image contrast. UV light is capable of causing minerals (e.g. bone [hydroxyapatite]) to fluoresce, and can even highlight soft tissue to some extent [7]. This paper describes a next-generation method of fluorescing minerals using specific wavelengths of light produced by a laser and corresponding imaging through the use of laser-blocking longpass camera filters (see Methods). This method is herein named Laser-stimulated fluorescence (LSF). For many decades UV light has been used at night to find and collect fluorescent mineral specimens, which are prized for their wide variation in color [8]. The field of biology has made tremendous scientific advances through the use of laser-induced fluorescence mostly through the widespread use of confocal laser microscopes [9–12]. In paleontology, UV light has seen increasing use in recent years where the resulting fluorescence can often reveal structures and patterns not seen under white light [13, 14]. The typical UV light source consists of commonly available standard fluorescent lamps with low wattage and a wavelength of 364 nanometers (nm) [7]. Greater amounts of UV flux on the specimen will cause fluorescent minerals to become more conspicuous, allowing for easier photographic documentation, sometimes with the aid of UV filters (e.g. Hoya brand) [6]. The limited variety of detectable fluorescence in fossils has been a primary limitation in the past using standard UV bulbs [14]. The technique presented here utilizes laser illumination to stimulate fluorescence which offers an order of magnitude improvement in the signal-to-noise ratio over standard UV light. The irradiance of a 20 watt UV fluorescent lamp is about 510 milliwatts per square centimeter (mWcm-2) at a distance of 20 centimeters from the target [15], but the irradiance of a ½ watt laser is on the order of 4000–8000 mWcm-2 [16]. This results in detectable fluorescence of many hard-to-fluoresce mineral types which typically remain dark under standard UV. This advantage can be leveraged when other factors are accounted for. For instance, matching the correct laser line with one of the specimen’s absorption bands provides more effective excitation of the fluorescence in a sample. Furthermore, using the right optical filter that matches one of the fluorescence bands of the specimen would improve contrast in the fluorescence image. Each color of laser emits a different wavelength of light, which will excite fossils and matrix from different rock units in different ways, as the case histories that follow will indicate. Again, LSF techniques depend on the wavelength of light used, the filter used, and the inherent fluorescent properties of the rocks under study. The exact methodology used, therefore, is going to vary depending on these properties. Laser-induced fluorescence imaging performed through confocal laser-scanning microscopy (CLSM) has been used in micropaleontology to study the morphology and cellular anatomy of fossils in situ, at micron-scale resolution, and even in three-dimensions [17–19]. LSF is a simplified and more accessible version of CLSM that uses simpler laser beam scanning and data acquisition systems, and lacks a confocal hole. However, the LSF technique provides its own unique advantages in studying macroscopic paleontological specimens including the compactness and low cost of its setup, its fast data acquisition rate and its high sensitivity compared to UV-stimulated fluorescence. The purpose of this paper is to describe the laser-stimulated fluorescence (LSF) imaging technique and to formalize its use in paleontology in the hope that new and more efficient modes of discovery will be possible.

Methods Laser-stimulated fluorescence (LSF) imaging is a versatile observational technique that has a multitude of paleontological applications. Both automated and manual systems can be used to scan or otherwise observe fossils under laser illumination. A series of common steps apply to any LSF work, these are detailed below. Laser light is concentrated on a specimen either as a point source for microscopic work, as a divergent light cone for smaller-sized specimens (with the aid of a laser diffuser), or a collimated beam (in which all light rays are parallel) is raster scanned over very large specimens. Since the laser is very bright, it must be blocked with an appropriate filter that still allows the longer wave fluorescence signal to pass through. Proper precautions using laser-blocking protective glasses and manufacturer’s safety protocol should be followed. The equipment used with this methodology depends on the exact wavelength of light produced by the laser. Specialized light-blocking longpass filters, often used in astronomy, are best-suited for these methods. These particular filters will allow all wavelengths of light longer than a certain wavelength to pass through the filter, however, it will stop all shorter wavelengths. For instance, a red-orange longpass filter (LP580, MidOpt) will allow 91–95% of light between the wavelengths of 600–1100 nm, however, the transmission sharply decreases between 600–520 nm, and by 510 nm, no light passes through the filter (www.midopt.com). A 477 nm blue laser would be efficiently blocked by this filter, but will still allow imaging of longer fluorescent wavelengths. The laser wavelengths and filters for each particular specimen were chosen experimentally via the trial and error method, a procedure that we believe is reasonable given the simplicity of this technique. The setup parameters for each of the five case histories presented in this study are given in Table 1. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Setup parameters for five case histories. https://doi.org/10.1371/journal.pone.0125923.t001 Standard UV bulbs can be used in addition to lasers in order to cover a broad range of the light spectrum. Imaging is done in both UVA from 315–400 nm and UVB from 280–315 nm. When working with UV light, photographs can be taken both with and without filters due to the low UV sensitivity of digital camera CCD (charge-coupled device) chips. No special digital cameras are needed to photograph specimens using laser fluorescence. Typically, digital single lens reflex cameras (DSLRs) capable of manual time exposures (e.g. Nikon D610) with either wide angle or macro lenses are sufficient. Ideally, the photography should be done in a darkroom, basement, or office without windows or with blackout curtains, as any influence of natural light will reduce the clarity of the fluorescence. The use of a tripod is necessary, as the exposure time during photography is typically long—up to several minutes, although this may not be the case for close macro photography. The aperture setting (f-value) should be as low as possible for long-exposure shots. Multiple types of laser light sources can be used. The more powerful the laser, the better and brighter the fluorescence. For the experiments outlined here, class III lasers in the 300–500 mW category were used. These were well below the threshold that results in radiation damage to the specimens studied. A lab laser, which plugs into the wall and is fairly static, and a high-powered laser pointer that runs off of CR123A lithium batteries, have both been used successfully depending on the locality of the specimen. The benefit of using a lab laser is that it can be used for hours at a time without overheating. It is typically used for precision work and photographing larger specimens. A high-power laser pointer is more portable and adjustable than a lab laser, however it can only be used for ~5 minutes, or else it will overheat and become damaged. If the photographer knows what f-value and shutter speeds are necessary for photography, a laser pointer can be used to great effect. It is excellent for macro photography in the field due to its portability. A laboratory setup for table-top-sized specimens would typically hold the laser on a fixed mount (Fig 1). The laser itself emits a collimated beam, which results in only a small dot of illumination. This beam can be used as is for maximum flux or be expanded using a diffuser (ARF used a 20-degree diffraction diffuser from Thorlabs). The smaller the angle of the diffuser, the better—i.e. 20 degrees would be better than 50 degrees, as it restricts the beam to a narrower angle and results in a brighter and smaller area of illumination, even if the laser is placed further away from the specimen. The laser should illuminate as much of the specimen as possible, and the diffuser’s cone angle changes the area covered by the laser depending on the laser-to-specimen distance. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Laser mounted on a stereo microscope. A 532 nm green laser mounted to the side of a stereo microscope. The blocking filter is mounted in front of the objective lens. No diffuser is used here for maximum effect with a smaller spot. https://doi.org/10.1371/journal.pone.0125923.g001 Larger specimens can be scanned using a custom device (Fig 2A and 2B). A Powel laser line lens projects a laser line in the Y direction that evenly distributes the laser energy over the length of the line (Fig 2C). A motor scans the entire assembly in the X direction (Fig 2A and 2B). This allows specimens of almost any size to be imaged. The exposure time of the DSLR camera should cover one or more of the X direction scans of the specimen. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Laser scanning system. A, Custom-built laser scanning device. B, Blue and green laser modules are mounted onto the scanning plate. A variable speed DC motor scans the plate back and forth laterally and is adjustable for degrees covered. C, Removable line lenses convert the laser spot to a more diffuse vertical line. Scale bar in Fig 2A equals 15 cm. https://doi.org/10.1371/journal.pone.0125923.g002 For a microscope setup, the collimated laser beam is directed through one of the illumination ports or projected directly onto the specimen. The emitted light, laser and fluorescence, comes back through the microscope’s optical train where a longpass filter is placed either before the objective lens or internally in a filter slot to block the intense laser light. The fluorescence can then be observed and photographed in detail. Specimen sources for each case history: Case history 1: Burke Museum of Natural History and Culture, UWBM 103073—feather from Green River Fm.; UWBM 103074—feather from Parachute Member of Green River Fm. Case history 2: Department of Land and Resources of Liaoning Province, LVH 0026—fish specimen from Jiufotang Fm. [20] Case history 3: UWBM 103075—microfossils from Brule Fm.; UWBM 103076—microfossils from Hell Creek Fm. Case history 4: Gobero specimen housed in the University of Chicago Research Collection, G1B2—juvenile female skeleton from mid-Holocene lake deposits Case history 5: Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China, IVPP V13320—Microraptor skull from Jiufotang Fm. [21]

Discussion There is a short list of non-destructive techniques that are both affordable and accessible for use in paleontology. Laser-stimulated fluorescence (LSF) is a new addition to this list. It provides an instantaneous, non-invasive, geochemical fingerprint of fossilized bone, soft tissue, integument and the surrounding matrix. LSF is also valuable for uncovering anomalies in specimens that direct the user towards more detailed investigations using other methods such as Raman spectroscopy—LSF is not presently quantifiable as to the precise elemental or molecular nature of the material. In addition to the aforementioned fossilized structures, anomalies can also relate to the use of glue, differences in matrix and bone composition, and the potential presence of composite skeletons. With higher sensitivity than UV lamp-based fluorescence imaging, and the compactness and low cost of the setup, LSF promises to become a diagnostic paleontological technique for everyday use. LSF imaging therefore has great potential for use in future descriptive work, particularly of fossils from well-preserved assemblages (konservat-lagerstätten) like those of the Mesozoic of northeastern China, which can have incomplete provenance information. In the examples given in this paper, new information was revealed using relatively inexpensive and rapid techniques that would otherwise have not been discovered without the use of much more expensive and/or time-intensive methods. Setups start from around US$500 and process within seconds to minutes making LSF easily accessible. The ability to look into the matrix for hidden specimens with simple techniques like the ones described here (Case history 2), was previously only possible using X-rays, CT scans, CLSM and RAMAN which have high cost and low accessibility [18, 23]. Micro-CT has certainly revolutionized the way small specimens are dealt with, but in virtually all cases the specimens are already known and CT is not likely to be used as a basic discovery process due to its expense. Silicates can often diffuse a concentrated laser beam several millimeters into the matrix. Future experiments may quantify the depth of matrix penetration using more powerful lasers and may extend the detection depth further than expected. This technique is particularly useful for fossils that are too large to fit onto a standard microscope stage or into regular CT scanners. It is also useful for fossils with an insufficient density contrast with their host matrix, which cannot produce meaningful CT images. The technique can therefore help to bridge a number of important knowledge gaps relating to such fossils. LSF can also be used in a novel way as a light source behind non-fluorescing features, such as carbon films (Figs 3 and 4). These soft-tissue remains have become progressively more important and are improving our understanding of fossil morphologies, but the techniques to investigate them have been slow to develop. Small details in carbon films can be missed using reflective light microscopy, however, the use of LSF will often backlight carbonized films both on and slightly below the surface. This clarifies the image and creates extra contrast that makes carbonized structures easier to see, especially smaller structures (e.g. the feather barbules in Figs 3 and 4). If LSF is adopted as a standard investigative tool, we may be able to further investigate carbonized films and other non-fluorescing soft tissues. The backlighting technique can also highlight material defects, which could be phylogenetically informative e.g. the distinctive cracking pattern of hippo ivory (Case history 4). Such defects may also reveal useful biomechanical and behavioral information if they relate to tooth, tusk and bone loading and wear. Multi-spectral imaging with different wavelengths highlights the fact that various minerals and compounds fluoresce with different intensity, according to wavelength. This is an important property as it may not always be desirable to fluoresce the entire specimen. In the case of the auto-picker, only bone specimens were of interest and the green laser was an ideal choice. Had a method such as UVB been employed the bone fragments would have been overwhelmed by other common minerals and organics, which also fluoresce under UVB, defeating the purpose of the machine.

Conclusions The advent of low cost, high power lasers including blue, green and violet wavelengths, means that the laser-stimulated fluorescence (LSF) technique can become widely available. The filters can be sourced from the photography industry or astronomy suppliers again at reasonable cost. The compactness and portability of the system combined with its efficiency in scanning large and extremely small fossils fills a gap in our ability to quickly and easily analyze specimens. It provides instant geochemical comparisons over a wide range of specimens, including specimens that previously exhibited very low fluorescence activity. At the microscopic level, LSF has already proven its worth in revealing unseen details in many specimens. The use of fluorescence is already in common use in multiple subdisciplines of biology, and is clearly a technique that is useful in paleontology as well. We have only scratched the surface of the ability of fluorescence to contribute to paleontology. LSF already holds a ‘first’ in the automation of micro-fossil sorting and this could theoretically be extended into automated preparation of specimens which is the single biggest bottleneck in paleontology. Overall, LSF is a discovery process geared toward uncovering unusual features of interest for follow up examination with more specialized tools.

Acknowledgments The authors wish to thank Peter and Debra Ceravolo, Chris Lively, Bob Davis and Zheng Wenjie. Craig Byron and an anonymous reviewer are thanked for their feedback, which resulted in significant improvements to this manuscript.

Author Contributions Conceived and designed the experiments: TGK. Performed the experiments: TGK ARF MP. Analyzed the data: TGK ARF MP PS LM DAB XX. Contributed reagents/materials/analysis tools: TK ARF LM DAB PS EG XX YW. Wrote the paper: TGK ARF MP.