For revising and studying plant inclusions from Baltic amber, holotypes and further original specimens described and published by Goeppert and Berendt (1845), Goeppert (1853), Menge (1858), Goeppert and Menge (1883), Caspary (1872a, b, 1886), Conwentz (1886a, b, 1890), and Caspary and Klebs (1907) were reinvestigated and evaluated. Although their collections have been preserved over the last centuries, they are incomplete since many specimens were lost during World War II, including several type specimens (Kosmowska-Ceranowicz 1990).
In order to find holotypes of plant inclusions and non-described specimens from Baltic amber, several historic amber collections were screened. Private amber collections including recently found amber pieces provided by several private collectors were also used in this study (see Tab. 4 for an extensive list). For revising holotypes whose whereabouts are unknown, historic literature (cited above), which supplied detailed descriptions and illustrations of the respective specimen, was used.
Tab. 4: Baltic amber collections examined for this thesis.
Name of collection Institution
Königsberg Amber Collection Geoscientific Museum, Geowissenschaftliches Zentrum (GZG), University of Göttingen
Hoffeins Amber Collection Geoscientific Museum, Geowissenschaftliches Zentrum (GZG), University of Göttingen
Berendt Amber Collection Museum für Naturkunde Berlin (MB), Germany Künow Amber Collection Museum für Naturkunde Berlin (MB), Germany
Carsten Gröhn Amber Collection Glinde; in parts at the Geological-Palaeontological Institute and Museum of the University of Hamburg (GPIH)
Jürgen Velten Amber Collection Idstein, Germany
Jörg Wunderlich Amber Collection Hirschberg an der Bergstraße, Germany
3.2 Preparation, microscopy and imaging
The majority of specimens used in studies involved in this thesis had already been prepared in the past by the respective collector(s) and/or former curators. However, some specimens showed scratches and fissures that cause light diffraction which does not allow an optimal visualization of the inclusions. For this reason, the respective amber specimens were carefully ground manually by using wet silicon carbide papers of different grit sizes (Struers company). The grinding was conducted in stages (500-800-1200-2400 grits) and terminated with a final polish, using a 4000-grit carbide paper and a leather polishing cloth with a tooth paste suspension (abrasive tooth pastes, e.g. Blend-a-med classic or Colgate). The ground facet had to be smooth and parallel orientated to the inclusion for an optimal view on its morphological details [see Nascimbene and Silverstein (2000) for detailed protocols on the grinding and polishing procedures]. For specimens that were embedded into high-grade epoxy resin (see chapter 3.3) a grinding machine (Buehler Eco Met 250) was used to create a smooth and even facet. The mechanical grinding procedure was
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the same as already described for manual grinding; however, the grit sizes were different, ranging from 80-320-600-1200-2500 grits. The final polish was conducted with a polishing cloth (VerduTex, Buehler) and a polycrystalline diamond suspension (MetaDi Supreme, Buehler). The polishing was done in stages, starting with a procrystalline diamond particle size of 3 µm and terminating with 1 µm particle size.
Each specimen was placed on an object slide and topped by a drop of water and a coverslip. The specimens were examined with Carl Zeiss microscopes (stereo microscopes Stemi 508 and Stereo Discovery V8, and a compound microscope AxioScope A1), using incident and transmitted light simultaneously. Inclusions were photographed using Canon EOS 5D digital cameras installed on each microscope. To accommodate the three-dimensionality of the inclusions, the software package HeliconFocus 6.0 was used to digitally stack the individual focal planes (up to 120 single images) in to one photomicrographic composite. For overview images of large specimens, up to four photomicrographic composites of the respective specimen were merged, applying the Adobe Photoshop CS6 software. Using a micrometer eyepiece, important morphological characteristics of each specimen were measured.
3.3 Permanent preparation
Some amber specimens exhibited deep fissures and cracks that extended to the inclusion. This facilitates deterioration of the amber inclusions, destabilizes the entire specimen and also impairs the optimal view of the inclusion (Nascimbene and Silverstein 2000, Pastorelli 2009, Bisulca et al. 2012). To stabilize amber specimens and to fill deep fissures, some specimens were embedded in a mixture of high-grade Epoxy resin (EPO-TEK 301-2, Part A) and hardener (EPO-TEK 301-2, Part B), following in parts the protocol by Nascimbene and Silverstein (2000). Before mixing both components, they were stirred beforehand to re-disperse settled particles. 17.5 g of Epoxy resin was weighed out and placed in a small plastic container (volume 4 cl); 7 g of hardener was added and both components were mixed with a glass rod until striations disappeared. If numerous air bubbles occurred during the mixing process, the air was released by placing the container for a short time into a vacuum drying oven (VO 200, with pump module PM 200, Memmert company) until a vacuum of 50 mbar was reached.
Meanwhile, each amber specimen was glued into a chamber of silicon ice cube trays (Lurch company), using Epoxy-Minute Adhesive (Weicon company). This is a fast-curing transparent epoxy resin with double cartridges, containing adhesive resin and hardener. After rejecting the first amounts of the double syringe on a mixing pad (Omnident company), both components were mixed thoroughly with a wooden pick.
Very small drops of this mixture were applied into the silicon moulds to attach the specimen to the bottom. This prevents the ‘floating up’ of the specimens during the embedding process. The Epoxy-Minute Adhesive needs to cure for about 30 minutes for a handling strength of approximately 35 %.
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Under a fume hood and using a plastic pipette, the epoxy resin-hardener mix was added to each chamber containing a specimen, covering the entire amber piece at least 1-2 mm above the upper facet. Then, the silicon mould was set into the vacuum chamber (adjust vacuum to 50 mb) and remained there for at least 15 minutes to ensure that the epoxy permeated the entire amber specimen. Air bubbles which remained in the epoxy resin were removed with a needle afterwards. For curing, the mould, including all embedded specimens, was placed into a fume hood for at least 3 days. Later, specimens were ground and polished, as described in chapter 3.2.
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4. Plant inclusions: their contribution to the understanding of vegetation,