Laser-stimulated uorescence reveals unseen details in fossils from the Solnhofen Limestone (Upper Jurassic, Bavaria, Germany)

Laser-Stimulated Fluorescence (LSF) has seen increased use in palaeontological investigations in recent years. The method uses the high ux of laser light to reveal details sometimes missed by ultraviolet (UV) and optical wavelengths. In this study, we compare the results of LSF with UV on a range of fossils from the Upper Jurassic Solnhofen Limestone Konservat-Lagerstätte of Bavaria, Germany. The methodology follows previous protocols with modications made to enhance laser beam intensity. Our experiments show the value of LSF in revealing shallow subsurface detail of specimens, previously not widely applied to Solnhofen fossils. In particular, fossil decapods from the Solnhofen Limestone reveal full body outlines, even under the matrix, along with details of segmentation within the appendages such as limbs and antennae. The results indicate that LSF can be used on both vertebrate and invertebrate fossils and may surpass the information provided by traditional UV methods in some specimens.


Introduction
Since the introduction of ultraviolet (UV) uorescence for the analysis of fossils from the Upper Jurassic Solnhofen Limestone lithographic limestones of Germany in the early 20th century (Miethe & Born 1928), the technique has been increasingly used in the analysis of exceptionally preserved fossils from this famous fossil Lagerstätte. Fossils from a wide range of phyla have been studied using UV including decapods (Schwiegert 2011), ammonites (Keupp 2007), sh ( (Fig. 1). The Solnhofen Limestone is famous for its well-bedded, ultra ne-grained lithographic limestones (often called plattenkalk) and referred to as such herein) that formed in the calm basins of the Solnhofen lagoons on the northern margin of the Tethys Ocean (Viohl 1998;Munnecke et al. 2008). The palaeoenvironment represented by these limestones is a closed lagoonal system with high evaporation rates leading to a strati ed water column with anoxic bottom waters largely devoid of macroorganisms (Viohl 1998). Occasional mixing through storms brought the toxic water to the aerated surface zone leading to the mass mortality of nektonic organisms (Pan et al. 2019). These organisms often became exceptionally well preserved due to a lack of scavenging, bacterial sealing, and rapid burial (Wellnhofer 2009). Here we compare images of Solnhofen fossils under LSF and UV and evaluate the use of LSF on fossils in this important Konservat-Lagerstätte.

Methods
The specimens used in this study labelled LB 1-13, abbreviated from the initials of the primary author, were collected during a series of eld visits to the Solnhofen region over twenty years and are accessioned in the collection of the School of the Environment, Geography and Geosciences (SEGG), University of Portsmouth. Additional specimens from the Staatliche naturwissenschaftliche Sammlungen Bayerns, Bayerische Staatssammlung für Paläontologie und Geologie (SNSB-BSPG) were studied by MP and TGK during a visit to the Museum für Naturkunde (Kaye et al. 2019a) (Figs 13-16). The specimens used are based on availability and represent some of the main groups found in the Solnhofen Limestones.

Photography
Photographs were taken in a blacked-out room to avoid natural light contamination. An LED lamp illuminated the specimen obliquely (c. 45 degrees) or directly for the white light photographs.
The method of Laser-Stimulated Fluorescence modi ed from Kaye et al. (2015) used an MGL-III-532-1~300mW green diode-pumped solid-state (DPSS) laser with a PSU-III-LCD power supply with a set output of 85mW. Alterations to the method included mounting the laser onto a camera track where only a trucking motion was permitted, so the 532 nm green laser moved through the x-axis to scan the specimen whilst maintaining the same perpendicular relationship (Fig. 2). Through substituting trucking for the panning motion in previous publications, the laser module maintains a constant distance from the specimen and therefore a constant beam intensity across the entire fossil. For Figs 13-16 the same methodology was used in Kaye et al. 2019a with an abbreviated method stated here. A 1W 405nm blue laser was used to induce uorescence and a Nikon DSLR was used to take the photographs with a 425nm blocking lter. Post-processing (equalisation, saturation, and colour balance) was then performed in Photoshop CS6.
The method of ultraviolet uorescence consisted of a 365 nm lamp as used by Tischlinger & Frey (2002) with the specimen illuminated as close as possible.
The long-exposures for each image under all three lighting regimes were taken using a Nikon D5300 DSLR camera mounted on a tripod with a 2-second self-timer setting that prevents camera movement from affecting the image. Aperture priority mode controlled the length of exposure following the method described by Eklund et al. (2018). They suggested a 10 second exposure for UV and the ISO was adjusted accordingly. Using a low ISO prevents grainy photos and with a small aperture, other light sources are prevented from contaminating the image. 30 second exposures were used for imaging under LSF with the ISO left at 100. The position and distance of the specimen from the camera remained constant for each method so that LSF, white light, and UV images could be collected e ciently. This lighting sequence allowed the O56 blocking lter that prevents camera over-saturation with LSF to be applied effectively.
This lter was removed for white light and UV photography, as although the lter can pick up UV uorescence, the uorescence is clearer without a blocking lter. Due to the attened nature of the fossils within Solnhofen laminites, repetitive photography and photo stacking techniques were unnecessary.

Health and Safety
It is important that when using LSF or UV techniques, appropriate safety precautions are observed. The green laser and UV lamp require laser and UV blocking goggles during operation and these methods were conducted in a locked room with a suitable exterior notice to prevent people from being harmed. Humans are most sensitive to green laser light (Galang et al. 2010) and by following these guidelines, the operators and others can be safeguarded. When using UV, 10-minute breaks were taken every 10-30 minutes to prevent eye damage and headaches (Tischlinger & Arratia 2013). Since pale colours uoresce under UV light, dark clothing was worn while conducting scans.

Cephalopoda
Cephalopods in the form of ammonites, belemnites and teuthoids occur frequently in the Solnhofen Limestone and are sometimes exceptionally well preserved (Fuchs et al. 2015). Many have been reported with aspects of their soft tissues preserved, including impressions of tentacles with hooklets in belemnites and teuthoids (Klug et al. 2016), the musculature of the mantle in teuthoids (Klug et al. 2015) and the siphuncle and pellicula in ammonites (Keupp 2007). Although ammonites make up a large portion of the fossils from the Solnhofen plattenkalks, they are often poorly preserved due to the aragonitic composition of the shell which is readily dissolved during diagenesis (Seilacher et al. 1976). This dissolution leaves behind an external or composite mould in the matrix, occasionally with the original outline and the calcitic aptychi in the body chamber (Keupp 2007).
Although rarely preserved, the original shell can be observed once replaced by calcite in the phragmacone (Fig. 4) and the body chamber (Arratia et al. 2015). The body chamber is present within the halo of the dissolved shell and can be enhanced under UV and LSF (Fig. 6). The siphuncle, pellicula and other nonmineralised elements are often phosphatised in Solnhofen ammonites and these may uoresce more intensely than the remaining shell (Keupp 2007). UV uorescence displays colour differences on these ammonites (Fig. 6C), but the contrast between non-mineralised parts and the surrounding shell is lacking, when compared with the LSF image of specimen LB 4 (Fig. 6D). It appears that the raised darker areas on isolated aptychi (LB 1) (Fig. 3) are the thick spongy layer on the inside of the aptychus underlying the thinner crenulated outer layer, rather than soft tissues following Lehmann (1976).
Lumbricaria Goldfuss, 1831, a coprolite attributed to ammonites (Janicke 1970) lies on the same slab as an ammonite (Fig. 6) and appears to contain aptychi of a smaller ammonite. Plesioteuthis prisca (Rüppell, 1829), a squid from the Solnhofen Limestone with a central rachis that is revealed in its entirety and uoresces at two distinct levels under LSF (Fig. 7D). This rachis runs along the centre of the visible body outline, although no soft tissues of the mantle are present (Fig. 7). be prepared further to assist the use of this method. Ultraviolet uorescence on a specimen of Alcmonacaris winkleri Polz, 2008 (LB 9) reveals a faint outline of the animal while recording colour patterning (Fig. 12). Under LSF, green laser light, this colour information is lost but the animal is revealed in its entirety (Fig. 12D). Techniques to rectify this loss of information have been developed using multiple wavelengths (Kaye et al. 2015). As Figs 9-12 show, the preserved exoskeleton and the body outline uoresce at different levels because of the auto uorescent compounds within the arthropod exoskeleton.

Vertebrata
The Solnhofen Limestone has achieved much of its fame as a fossil Konservat-Lagerstätten because of the exceptional preservation of its vertebrate fossils, especially those of volant animals such as the earliest unequivocal fossil bird Archaeopteryx and a diverse assemblage of pterosaurs ( (Figs 13-16). The hollow bones of pterosaurs are rarely preserved and through LSF the contrast between preserved bone and the imprints of the skeleton in the matrix is exempli ed. The dwarf crocodilyform Alligatorellus (Fig. 16) under the blue laser displays soft tissues around the entire body as well as a brighter section at the base of the tail that may represent partial preservation of the caudofemoralis muscle.
However, the exceptional preservation is not restricted to the Tetrapoda, with many shes also exceptionally well preserved with full articulation and soft part preservation ( (Fig. 19), correspond with previous studies using UV for uorescence (Tischlinger & Arratia 2013).
Fish uoresce well under both UV and LSF and although the difference is often minimal, clearly in Fig. 18 LSF surpasses the UV photograph with an increased uorescence of both the skull and vertebral column completing the structures seen and this results from the higher ux and subsurface illumination.

Costs
With reductions to the cost of laser systems, the method could be replicated using a 532nm laser, an LCD power supply and a Zecti 31.5in"/80cm camera slider. This study using an 85mW laser shows the technique can be used with less powerful laser equipment than the 300-500mW laser used by

Conclusions
The list of non-destructive techniques available to palaeontologists is increasing. X-rays were rst implemented in 1896 on fossils from another German Lagerstätte, the Devonian Hunsrück Slate        showing that the central rachis is raised (C). LSF using the 532 nm laser uoresces this gladius at different levels with the central vane picked out through its higher uorescence. Scale bar = 10mm.    above with the addition of the antennular peduncle (ped) from Audo and Charbonnier (2012). Scale bar = 10mm. B and E magni ed x2 and C and F magni ed x3.6.