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J. Paleont., 77(6), 2003, pp. 1182–1192Copyright q 2003, The Paleontological Society0022-3360/03/0077-1182$03.00

NEW APPLICATIONS OF LIGHT AND ELECTRON MICROSCOPICTECHNIQUES FOR THE STUDY OF MICROBIOLOGICAL

INCLUSIONS IN AMBERCARMEN ASCASO,1 JACEK WIERZCHOS,2 J. CARMELO CORRAL,3 RAFAEL LOPEZ,3 AND JESUS ALONSO3

1Centro de Ciencias Medioambientales, CSIC, Serrano 115 bis, 28006 Madrid, Spain, ,ascaso@ccma.csic.es.2Servicio de Microscopıa Electronica, Universidad de Lleida, Rovira Roure 44, 25198 Lleida, Spain,

,jacekw@suic-me.udl.es., and3Museo de Ciencias Naturales de Alava. C/Siervas de Jesus, 24. 01001 Vitoria-Gasteiz, Spain,

,mcna@jet.es.

ABSTRACT—Amber is a superb medium for the fossilization of delicate organisms. Besides light microscopy techniques for the studyof insects in amber, scanning electron microscopy (SEM) in backscattered electron mode (SEM-BSE), low temperature SEM (LTSEM)and also confocal laser scanning microscopy (CLSM) were used to examine microcenosis and particulate plant remains (microdebris).We applied these techniques to such inclusions in amber Alava, northern Spain (Allaian: Early Cretaceous). Confocal microscopyprovides a 3D image of partial microcenosis showing bifurcate fungal hyphae. The huge potential of SEM-BSE yields high resolutionimages, in which the relationship between protozoa and fungal hyphae may be observed and the characterization of further ultrastructuraldetails in flagellates. According to the SEM-BSE images, food and pulsatile vacuoles appear better preserved than mitochondria andlipids in amber-embedded protozoa. A process of protozoan mineralization has led to the deposition of S and Fe in peripheral areas,and the Fe is also present in the core of surrounding fungal hyphae. Application of LTSEM for the study of protozoan inclusionsproduces images of their exteriors showing many vacuoles. Plant tissues under SEM-BSE show mummified cell walls, while thecytoplasm exhibits a bright appearance and is very rich in Fe and S. SEM in secondary electron mode (SEM-SE) also reveals amicrobiota trapped in gas bubbles.

DIAGRAM 1—Schematic representation of the preparation proceduresused for SEM-SE performed at room temperature and LT-SEM at lowtemperature.

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FIGURE 1—1, Light microscope (LM) image of polished sample of amberfrom Penacerrada (Alava, Spain), showing fungal hyphae and protozoa;2, confocal laser scanning microscopy (CLSM) 3D image of the sameamber microcenosis showed in 1 (at higher magnification), also show-ing protozoa and bifurcate hyphae. The insert 3, shows a detailed imageof a protozoa, based on a single confocal section; 4, same area as in2, but wider field. Scanning electron microscopy in backscattered elec-tron mode (SEM-BSE) image. The zone previously examined byCLSM in 2 is marked by an asterisk. In the lower part of the figure isa mixture of fungi and protozoa; 5, SEM-BSE image, showing intimateproximity of protozoa (arrows) and fungi; 6, SEM-BSE image. Fla-gellate protozoa showing flagellar pocket (open arrow), anterior fla-gellum (curved arrow), and recurrent flagellum (broken arrow). Recur-rent flagellum lies in contact with cellular body (arrow heads). Fungalcells (thin arrows) appear in different sectional planes; 7, SEM-BSEimage shows a hyphal strand in longitudinal section with a central core(arrows) and radial filaments; 8, transverse section of a hyphal strand,exposing the central core and radial filaments.

INTRODUCTION

FOSSIL RESINS in Spain occur mainly in Lower and middle Cre-taceous sedimentary deposits (Ramirez del Pozo and Aguilar,

1969; Aguilar et al., 1971; Cherchi and Schroeder, 1982; Wilm-sen, 1997), but generally only as traces, and lack fossil macroinclusions. Arbizu et al. (1999) have recently reported some insectinclusions from an amber site in Asturias (northern Spain) pre-viously studied by Casal (1762) who first mentioned several am-ber occurrences in the jet mines well-known at that time.

The amber from Alava is exceptional, both in geological andpalaeontological terms (see Alonso et al., 2000). It is one of only

two known highly fossiliferous amber deposits of Lower Creta-ceous age. The other deposits being from Lebanon (Schlee andDietrich, 1970; Azar et al., 1999a, 1999b). The detailed preser-vation of amber inclusions enables more precise taxonomic, phy-logenetic and palaeoecologic studies than those afforded by com-pression fossils. This is of particular interest in view of the spec-tacular adaptive radiation of angiosperms that occurred during thelate Lower and early Upper Cretaceous (Cenomanian-Santonian)(Crane et al., 1995).

Microcenose in other ambers have been found to harbor bac-teria. This has been shown by Poinar (1992) and Wier et al.(2002) using transmission electron microscopy (TEM), by Kohr-ing (1995) using scanning electron microscopy in secondary elec-tron mode (SEM-SE), and by Waggoner (1994) and Schonbornet al. (1999) using light microscopy (LM). Cano and Boruki(1995) have even purportedly revived and identified Bacillus bac-teria in amber from the Dominican Republic. Dominican amberhas been dated as 15 to 20 million years old (Miocene) based on

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FIGURE 3—1, LTSEM (SE detector) image showing a protozoan with many vacuoles; 2, LTSEM (BSE detector) image showing internal view of amineralized protozoan with several vacuoles (arrows).

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FIGURE 2—1, SEM-BSE image. Two flagellate protozoa surrounded by hyphae; 2, SEM-BSE image. Flagellate protozoan shown at higher magnifi-cation; 3, the center of the insert shows a pulsatile vacuole; 4, SEM-BSE image. Ciliated protozoa, perhaps of the genus Paramecium. Mouthappears as a channel (black arrow). Buttom of food channel indicated by white arrows; 5, SEM-BSE image showing an amoeba with severalpseudopodia; 6, SEM-BSE image. Amoeba with temporary extension (pseudopod) of its body (arrows); 7, SEM-BSE (EDS) image. Protozoasurrounded by hyphae. Images represent the spatial distribution of O, S and Fe.

fossilized foraminifera (Iturralde-Vincent and Macphee, 1996).More recently, Lambert et al. (1998) reported isolates of Staphy-lococcus in soil and plant fragments embedded in the amber itself.Viable bacterial spores in amber are highly controversial, sinceDNA itself is disputed to be preserved in amber (Smith and Aus-tin, 1997; Stankiewicz et al., 1998). Several genera of fungi havebeen described by different authors using LM. Rikkinen and Poin-ar (2001) reported fungal colonies fossilized in amber from theDominican Republic. Also described are protozoa (Poinar, 1992;Waggoner, 1994; Schonborn et al., 1999), invertebrates—mainlynematodes and arthropods (Poinar, 1992), insects (Poinar, 1992;Henwood, 1992a, 1992b), mosses and lichens (Poinar and Poinar,1999; Poinar et al., 2000), and plant fragments (e.g., Weitschatand Wichard, 1998). Poinar and Hess (1982), Poinar (1992) andGrimaldi et al. (1994) obtained transmission electron microscopy(TEM) images of insect tissues. SEM-SE was recently applied byGrimaldi et al. (2000b, 2000c) to the study of inclusions, mainlycomposed of wood, crystals and fragments of insects. Grimaldiet al. (2000a) also applied an interesting technique based on ultrahigh resolution X-ray computer tomography (UHR CT) on inter-nal features of macroinclusions in amber, such as the bones ofsmall vertebrates.

The aim of the present study was to analyze the microcenosisand microdebris in amber from Alava (Spain), dated 114 millionyears old, using methods with exceptional magnification and res-olution.

Geographic and geological setting.The amber deposits(Penacerrada-I and Penacerrada-II) are located approximately 30km south of the city of Vitoria-Gasteiz, near the village of Pena-cerrada. This mountainous region, known as the Sierra de Can-tabria (Alava), is part of the South Pyrenean Frontal Thrust, an

important geological structure facing the Neogene foreland Ebrobasin.

Yellowish clastic sequences from the Aptian to Cenomanianaccumulated in this region. According to micropalaeontologicaland sedimentological criteria, two formations can be recognized,the Nograro Formation (Aptian-Albian in age, Ramırez del Pozoand Aguilar, 1969) and younger Utrillas Formation (Upper Albianin age, Aguilar et al., 1971), the former with more marine influ-ence. The limit between these formations is often problematicsince uppermost strata of the Nograro Formation reflect a gradualtransition towards the continental clastic facies of the Utrillas For-mation. Most sedimentological information on the amber depositcomes from the best studied outcrop (Penacerrada-II) (see Alonsoet al., 2000). According to these authors amber was deposited inlow energy areas of a distal fluvial environment, where stagnantwaters allowed the formation of lignite and pyrite nodules. Therealso was a connection with marginal marshes or marginal bays ascorroborated by the presence of marine dinoflagellates. Barronand Elorza (2000) studied the palynology of the amber depositsand proposed a Middle Aptian-Upper Albian age for the amber.This age can be more precisely determined by the presence of awell defined magnetic reversal in the stratigraphic column, sug-gesting an Upper Aptian (chronozone M-1r) or possibly an earlyAptian age (chronozone M-0r) (Larrasoana and Garces, 2000).

MATERIALS AND METHODS

Collection and preparation.Amber was initially hand col-lected directly from the strata; subsequently a backhoe loader wasused to remove vegetation and soil in order to expose the mostinteresting levels. Several tons of amber-bearing black shales and

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organic-rich sandstones were thus extracted. As amber was scat-tered among these sediments, a concentration treatment was nec-essary to recover the amber lumps (see Corral et al., 1999).

The amber pieces were then carefully screened for inclusionsunder a light microscope. In most cases, amber pieces with inclu-sions were embedded in an epoxy resin (Epotek 301) to eliminatethe ‘‘mirror-effect’’ of internal cracks when illuminated, accordingto the method described by Schlee and Dietrich (1970) used forCretaceous ambers (e.g., Grimaldi et al., 1997; Corral et al., 1999;Nascimbene and Silverstein, 2000). This treatment also guaran-tees the conservation of amber prone to natural oxidation andeventual darkening. Finally, amber samples were trimmed andpolished for optimal observation of organisms. During this pro-cess, more than 1,500 biological inclusions—mostly insects—were isolated and catalogued (Alonso et al., 2000).

Amber is a superb medium for the fossilization of fragile or-ganisms, preserving even the most delicate structures of smallarthropods (Alonso et al., 2000). Recent studies on Alava amberconfirmed the presence of numerous biological inclusions, includ-ing many new species of insects (e.g., Arillo and Mostovski,1999; Arillo and Nel, 2000; Arillo and Subias, 2000; Baz andOrtuno, 2000, 2001a, 2001b; Szadziewski and Arillo, 1998; Wa-ters and Arillo, 1999), in addition to cryptogamic plant remainsand indeterminate leaf impressions preserved on the surface ofpieces of amber. A set of avian feathers have even been found,including a superb fragment of a contour feather.

Confocal laser scanning microscopy.Confocal laser scanningmicroscopy (CLSM) has been successfully applied to the studyof the geomicrobiological system (Rautureau et al., 1993; Ascasoet al., 1998; Wierzchos and Ascaso, 2001). This technique offersa novel opportunity for in situ study of organic features trappedwithin amber. These features were characterized by detecting au-tofluorescence of mummified microorganisms. It was observedthat some microdebris also showed fluorescence due to celluloseand other natural fluorocromos. For the CLSM study, the polishedblocks of amber were mounted on microscope slides using dou-ble-sided adhesive tape, and observed using a LSM 310 Zeissconfocal microscope with a Plan-Apochromat 633/1.40 oil im-mersion objective. An argon (488 nm) and helium/neon (543 nm)laser were used to generate an excitation beam; the resultant emis-sion was filtered through long pass filters of .515 nm and .575nm, respectively. The relatively good translucency of the amberpermits a three-dimensional (3D) reconstruction of fungal andprotozoan colonies and gives an idea of their spatial organization.To obtain this information, stacks of 20–30 single confocal opticalsection (vertical (z) resolution about 0.6 mm) images are preparedat 0.5–1 mm intervals through the sample and digitally stored andcompiled.

SEM-BSE and SEM-SE examination.The use of backscat-tered electrons in SEM yields high magnification images withcontrast attributable to differences on average atomic number ofthe target (Joy, 1991). For SEM-BSE observation and/or energydispersive X-ray spectroscopy (EDS) microanalysis, the amberfragments were embedded in epoxy resin. After polymerization,the blocks were cut and finely polished (Wierzchos and Ascaso,1994). Transverse sections of polished surfaces were carbon-coat-ed and examined using a DSM 940 A Zeiss and a DSM 960 AZeiss microscope (both equipped with a four-diode, semiconduc-tor BSE detector and a Link ISIS microanalytical EDS system).SEM-BSE and EDS examinations of the samples were done si-multaneously. The microscope operating conditions were as fol-lows: 08 tilt angle, 358 take-off angle, 15 kV acceleration poten-tial, 6 or 25 mm working distance and 1–5 nA specimen current.

Despite the advantages of the SEM-BSE technique over theSEM-SE method, the latter is also appropriate for the study ofmicrobiota in amber since the external morphology of various

microbes can be of interest because SE signals give contrast basedon topography. Prior to SEM-SE examination, amber sampleswere fractured in order to expose a clean, fresh surface, thencoated with gold.

LTSEM examination.Amber fragments were also examinedusing the LTSEM technique. Small fragments were mounted withO.C.T. compound (Gurr) and mechanically fixed onto the speci-men holder using the cryotransfer system (Oxford CT1500). Sam-ples were plunge-frozen in subcooled liquid nitrogen and thentransferred to the preparation unit. The frozen specimens werecryofractured and etched for two minutes at 2908C. After icesublimation, the etched surfaces were gold sputter coated (or insome cases only coated with carbon). Samples were subsequentlytransferred into the cold stage of the SEM chamber. Fracturedsurfaces were observed with a DSM960 Zeiss SEM microscopeat 21358C under conditions of a 15 kV acceleration potential, 10mm working distance and 5–10 nA probe current.

Comments.A secondary electron detector was used both onsamples fractured at room temperature (SEM-SE) and frozencryofractured samples (LTSEM-SE) (see Diagram 1). TheLTSEM technique is highly appropriate for analyzing the internaland external appearance of structures possibly containing watersuch as protozoa. In future studies, this method will be appliedto liquid-containing bubbles. The only difficulty with the use ofthis technique derives from the very small size of the samples tobe fractured. Once placed in the microscope’s prechamber, thesamples are fractured using a cooled blade tip and, although theprocess is visualized using a binocular magnifier, it is not alwayspossible to control the line of fracture.

RESULTS

Figure 1.1 shows a light microscope (LM) view of an area froma polished sample of amber from Penacerrada. Fungi and protozoamay be observed but the light microscope’s low magnificationdoes not provide detailed resolution of fungi and protists (whichcould be protozoa, slime moulds or microalgae). In Figure 1.2,confocal microscopy provides a 3D image of part of the samepiece of amber showing microcenosis with bifurcate fungal hy-phae. The 3-D images are reconstructed from a series of opticalsections. The fungi are not mineralized and the autofluorescenceprovides a sharp image. As far as we are aware, this is the firsttime that the CLSM has been applied to the study of microbiotain amber. The insert (Fig. 1.3), shows a detailed image (singleconfocal section) of a protozoan close to the hyphae.

The same area observed as a whole is shown in Figure 1.4, butin this case, examination was by SEM-BSE. In the figure is acentral channel (arrows), and in the lower part of the image mi-crobiota are apparent owing to their mineralization. The area pre-viously examined by CLSM (Fig. 1.2, 1.3) is indicated with anasterisk. No mineralized microorganisms were found in this area,but the presence of organisms with well-preserved autofluores-cence permitted the use of CLSM. Two zones may be discernedin Figure 1.4 in the lower half of each side of the figure containingmixtures of fungi and protozoa. This image was taken at lowmagnification to give a general view of the sample. The hugepotential of SEM-BSE yields the image at higher magnificationshown in Figure 1.5, in which the interrelation between protozoa(arrows) and fungal hyphae may be noted. Figure 1.6 shows aflagellate protozoan and a flagellar pocket in detail (open arrow).The flagellum inserted anteriorly is the weaker of the two (curvedarrow) and the recurrent flagellum is wider (broken arrow) andlies in contact with the cell body (arrow heads). This flagellate issurrounded by fungal hyphae (thin arrows), which appear in dif-ferent sectional planes each as a bright central core with surround-ing halo.

Figure 1.7 shows a detailed image of a hypha in longitudinal

1187ASCASO ET AL.—NEW MICROSCOPE TECHNIQUES FOR AMBER INCLUSIONS

FIGURE 4—1, SEM-BSE image. Mummified plant tissue showing cell walls. White grains indicate deposits of S and Fe (black arrows); 2, SEM-BSEimage. Detail of the grains in 1, revealing pyramidal crystals of pyrite; 3, SEM-BSE image. Mummified cell walls (black arrows) and mineralizedcytoplasm (white arrows); 4, SEM-BSE image. Area of contact between two plant cells. In the cytoplasm of the lower cell (white arrow) it ispossible to see a chloroplast (black arrow).

section. Note the central core (arrows) and an outer zone whereradial filaments occur. The image of a hypha in crossection (Fig.1.8) exposes the core 2 to 3 mm diameter and radial filaments ofthe exterior, which is 1–2 mm thick. Thus, hyphae that are opaqueto LM are bright inside to SEM-BSE. This indicates that the coreand radial structures of the envelope accumulated compoundswith a higher atomic number then surrounding matrix. EDS mi-croanalysis determined that the bright core and radii containedhigh S and Fe. In addition to the protozoan in Figure 1.6, severalother types were found, including amoebae, other flagellates and

ciliates. Figure 2.1 shows two flagellate protozoa surrounded byhyphae, like those in Figure 1.7 and 1.8. SEM-BSE at highermagnification reveals further ultrastructural details in the flagel-lates (Fig. 2.2). The inset (Fig. 2.3), taken at even greater mag-nification, shows a pulsatile vacuole in the center. Pulsatile vac-uoles of present-day protozoa observed by TEM (Cann, 1986)also show a halo of small vesicles. The remaining vacuoles seenin Figure 2.1 and 2.2, may be food vacuoles. According to theSEM-BSE images, food and pulsatile vacuoles seem to be betterpreserved than mitochondria and lipids in protozoan inclusions.

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FIGURE 5—1, SEM-SE image. Piece of amber with fractured bubbles. The insert 2 shows a bubble observed using the stereoscopic microscope; 3,SEM-SE image. General view of a bubble, showing the exterior of presumed microorganisms; 4, SEM-SE image. Mesh of filaments which couldbe a mummified bacterial colony; 5, detail of 4. 6, SEM-SE image from bubble wall. Spiroidal, coccoid and tape-like structures of presumedmummified bacteria; 7, detail of 6.

Figure 2.4 might correspond to a ciliated protozoan, perhaps ofthe genus Paramecium, which is a ciliated filter feeder. Its mouthappears as a channel (black arrow) and food is pushed towardsthe bottom of this gullet in the zone indicated by white arrows.Although the flagella of protozoa are clearly visible with SEM-BSE, this is not true of cilia. In the present study we were unableto observe cilia in any presumptive ciliate protozoan. Figure 2.5might represent a section of an amoeba with several pseudopodia.Figure 2.6 also shows a sectioned amoeba with a pseudopod ap-parent (arrows). This amoeba is surrounded by fungi like the otherprotozoa examined, but the separation between the amoeba’s cellmembrane and the fungi is much narrower than with flagellatesand ciliates.

Figure 2.7 shows a protozoon surrounded by hyphae, and fol-lowing images reveal the spatial distribution of O, S and Fe usingEDS. The mineralization process of protozoa led to the depositionof S and Fe in peripheral areas and Fe in the core of the sur-rounding fungal hyphae. Oxygen, preceded from iron hydroxides,was mainly restricted to the central zone of the protozoan whereFe may also be seen.

Application of LTSEM to protozoa produces images of theirexteriors showing many vacuoles. Figure 3.1 shows a flagellate(no cilia apparent) and the food vacuoles comprising it, perfectlycoinciding with that observed in the SEM-BSE images obtainedin polished amber in Figure 2.1 and 2.2. Image 3.1 was obtainedat low temperature with a secondary electron detector. When asample fractures during the LTSEM process, the interior of a min-eralized microorganism can be seen, as in Figure 3.2. For Figure3.2 the LTSEM image was obtained using a BSE detector, re-vealing several vacuoles (arrows), or areas of less BSE signal.The mineralized remains of this protozoan show enhanced bright-ness using EDS, due to its Fe-S composition.

Continuing with SEM-BSE applications, Figure 4.1 shows aremnant of plant tissue in which the cell margins is distinguishedowing to mummification of the cell walls. White stains occur inthe cell walls, indicated by white arrows (figure left), which aredeposits of S and Fe. On the right hand side of the same figurethere is a large amount of S and Fe, but this time in the form ofbipyramidal crystals (block arrows and Fig. 4.2). These pyritecrystals usually appear in the space between the walls of twoadjacent plant cells.

Figure 4.3 shows the spatial relationship among several plantcells, with a dark grey zone formed by the mummified walls(black arrows). The cytoplasm (white arrows) in many plant cellsoccupies a narrow space between the plasmalemma and the to-noplast of the central vacuole, is very rich in S and Fe, giving itits bright appearance. Here, the S and Fe do not form crystalsidentifiable with electron microscopy. Figure 4.4 shows the areaof contact between two plant cells. In the area of the cytoplasmof the lower cell (white arrow) it is possible to see a chloroplast(black arrow).

Finally, SEM allowed visualizing the microbiota trapped in thebubbles. Figure 5.1 shows a low magnification SEM-SE image ofa piece of amber with fractures through the bubbles. The inset(Fig. 5.2) shows the same area as in Figure 5.1 with sectionedbubbles, but observed using a stereoscopic microscope. The im-age shown in Figure 5.3 is a general view of the contents of abubble. As the fractured sample was handled at room temperatureand observed using SEM, it was possible to see only the exterior

morphology of the microorganisms, which prevents knowing theirtrue nature. Figure 5.4 (SEM-SE image) shows a tangled meshof filaments, which could be a mummified bacterial colony. Thismorphology is highly characteristic and resembles that of somegenera of ‘‘iron bacteria’’, such Gallionella. A detail of Figure5.4 is shown in Figure 5.5. Another bubble imaged with SEM-SE is illustrated in Figure 5.6, showing spiroidal (arrow), coc-coidal and tape-like structures. These are likely to be remains ofGallionela, with its twisted stalk morphology, and Laptothrixsheaths. Figure 5.7 is an enlargement of Figure 5.6 clearly show-ing coccoidal forms and perhaps dividing coccoidal bacterialcells. In this bubble, it was possible to see protozoa encrusted inits walls.

DISCUSSION

Some authors have considered, and even proposed dissolvingthe amber (Azar, 1997) to gain access to inclusions. In our opinionthis is unnecessary, even for the study of the smallest microor-ganisms, if high resolution techniques such as SEM are used.SEM may be performed both at room (conventional SEM) andlow temperature (LTSEM), using either a secondary (SEM-SE)or backscattered (SEM-BSE) electron detector. These methods arefurthermore the most appropriate in order to complement and no-tably improve conventional LM procedures. Schonborn et al.(1999) reported some interesting LM observations of a microcen-osis in amber, initially reported as Triassic but now consideredCenomanian in age (Schmidt et al., 2001). With the room tem-perature SEM secondary or backscattered electrons may be usedto obtain an image, which is of considerable significance for thestudy of amber. Conventional SEM-SE has yielded useful data forthe taxonomy of ciliated protozoa in aqueous media (Foissner,1991). Based on this technique, it has been possible to 3-dimen-sionally reconstruct the oral apparatus of ciliated protozoa (Hof-mann-Munz et al., 1990), but this invariably involved removingthe organisms from the aqueous medium in which they lived.SEM-SE can also provide fine structural details of macroscopicand relatively hard components included in the amber, such asinternal tissues and cuticular microsculpture of arthropods andwood (Grimaldi et al., 2000a, 2000b, 2000c). The method canalso provide interesting information about microscopic inclusionssuch as fungi or bacteria, as long as these are within empty cav-ities, not embedded directly in the amber (Fig. 5.3–5.7).

When we began our SEM investigations on the Alava amber,we were unaware of the stability of the resin to the electron beam.Poinar (1992) first addressed this problem while attempting toobserve bacteria using TEM of ultrathin sections of amber, whichunderwent severe deterioration. When a thin section is examinedusing TEM it is susceptible to the heating effect of the electronsand holes can appear. We nevertheless noted no substantial effectswhen observing thin sections of the Alava amber using the TEMat 80 kV (unpublished results).

In SEM-SE observations made on samples coated with goldaccording to standard procedures used by Grimaldi et al. (1994),Grimaldi (1996) and Grimaldi et al. (2000a, 2000b, 2000c), theamber maintained its integrity under the electron beam. The tech-nique proved useful for observing microscopic elements of woodand insect fragments, and microscopic elements with cell wallstrapped within bubbles. However, microcenoses commonly do not

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occur freely in a bubble or free space, but rather are fully em-bedded and surrounded by resin. Inclusions range from compo-nents of microcenoses, to other biological forms that are neitherunicellular nor microscopic, like remnants of plant and animaltissue. What we term microdebris, are fragments of macroscopicstructures that need to be identified with the aid of an electronmicroscope.

Our research team (Wierzchos and Ascaso, 1994) developed aprotocol for the study of microorganisms in lithic substrates. Inthe present case, this technique consists of observing a polishedamber surface or even a thin section such one used for LM, whereone expects to find microdebris. It should be noted that matricesthat appear cloudy in LM, can sometimes be clearly visible withSEM-BSE. Using SEM-BSE we tried to observe the componentsof microcenoses that lacked a cell wall, such as protozoa, as wellas organisms with cell walls such as bacteria, fungi and algaecompletely embedded in resin, as well as fragments of soft tissues.In this protocol, a sample of polished amber coated with carbonwas used, and behavior of the amber was ideal, with no damageor other heat alteration. The signal detected by this method showsthe difference in the mean atomic number of the structures ob-served (Joy, 1991). Thus, structures with a low atomic number(e.g., amber) appear as a shade of dark grey. Different shades ofdark gray are also shown by mummified plant and animal frag-ments and by dehydrated components of microbiota. However,some microorganisms are mineralized and have enhanced bright-ness. Areas of microdebris also show bright deposits. This samplepreparation method for SEM-BSE allows good observation of theentire microcenosis and the inner ultrastructure of all details.

Ultrastructural details observed in protozoa in amber, such ascontractile vacuoles (Fig. 2.2, 2.3), are of great use for the iden-tification of these microorganisms. Furthermore, given that resinrapidly traps organisms, it is a unique substrate for the preser-vation of symbiotic associations, particularly since ultrastructurecan aid in the investigation of symbiotic relationships.

Qualitative and/or quantitative analysis of chemical elements inthe inner structure of microorganisms (usually S and Fe), providesinformation on processes of biomineralization and fossilization.

When examining biological inclusions in amber, avoiding theloss of traces of water or liquid from the inside of some bubbles,LTSEM appears to be a good method of examining embeddedorganisms such as protozoa (Fig. 3.1, 3.2). The present work isthe first application of this technique for the study of protozoa inamber, and we believe that this method has not even been usedto examine recent protozoa. Thus, LTSEM seems highly prom-ising for exploring microinclusions with high water content suchas protozoa, and for analyzing bubbles containing liquid. TheLTSEM procedure shows that fungi abundant in the sample donot appear to be saprophytic protozoa. The close proximity be-tween fungi and protozoa (mainly shown in Fig. 1.5, 1.6, 2.2, 2.3,2.4, 2.6 and 2.7 obtained by SEM-BSE), suggests an intimateassociation between them, perhaps indicating that some protozoafeed from fungi. Mycelia shown by confocal microscopy are bi-furcate (Fig. 1.2), and do not appear to be epiphytic nor epixylicsince they were not found on plant remains. Inclusions of fungalhyphae observed by SEM-BSE appear to be largely coenocytic(see Fig. 1.7), allowing their assignment to specific groups offungi. Inclusions of plant fragments are very well preserved,probably because of dehydration since the cell walls of these tis-sues were mummified (Fig. 4.1–4.4).

Intercellular spaces and vascular elements have numerous crys-tals composed solely of Fe and S, which, also considering theiroctahedral morphology are pyrite crystals. Such pyritized micro-debris are usually a result of exposure of the inclusions to thesurface of amber via fine fractures, through which mineral-ladenwater can seep in. It is unknown what endogenous factors would

promote pyritization of fully sealed inclusions. When biologicalinclusions are highly mummified, SEM-BSE is the best methodto analyze these elements. Mummification may be commonly as-sociated with biomineralization, which would be important to ex-plore in future studies. Figure 4.3 shows the point of intersectionof four plant cells: adjacent walls were mummified but remnantsof cytoplasm from the four cells are mineralized. SEM-BSEshows both dehydrated walls in dark grey and protoplasts, whichowing to biomineralization have a higher atomic number and ap-pear lighter in color. Confocal laser microscopy provides a dif-ferent image of the non-mineralized biological parts (Fig. 1.2,1.3), since the method can detect fluorescence emitted from com-plex samples of cellulose or biomolecules of unknown composi-tion.

Coating the sample with gold and using only the topographicalinformation obtained with a secondary electron detector (SEM-SE) impairs distinction between organic and inorganic phases.However, visualization of the contents of bubbles using SEM-SE(Fig. 5.3–5.7) suggested the presence of a genus of bacteria com-monly found in fresh water, based on recognizable structures suchas twisted stalks (Gallionella) and sheaths (Leptothrix) (Ghiorseand Ehrlich, 1992). These bacteria produce extracellular structures(a stalk and sheath respectively) that become heavily encrustedwith iron oxides. Structures encrusted by iron-oxide remain longafter the bacterial cells dissipate, leaving empty tube-like sheaths.

Studies of amber using conventional SEM-SE, new methods ofSEM-BSE and low temperature scanning electron microscopy(LTSEM) can and should be complemented with confocal andlight microscopy. SEM-BSE and confocal microscopy can be per-formed on fairly thin sections and may help the taxonomic iden-tification of microbial inclusions, allowing a unique window tothe evolution of protists, particularly any preserved in older, Me-sozoic ambers.

ACKNOWLEDGMENTS

Foremost, we sincerely thank D. A. Grimaldi (New York) andG. O. Poinar, Jr. (Corvallis, OR) for their much appreciated sug-gestions and for improving the English of the original manuscript.We also thank F. Pinto, T. Carnota and S. Lapole for technicalassistance; A. Martın-Gonzalez and J. C. Guitierrez, M. SperanzaFernandez and M. J. Martınez for suggestions and A. Burton fortranslating the manuscript. The authors also thank W. Sanders.This study was funded by the projects: Proyecto Ambar-2000—Diputacion Foral de Alava and Proyecto BOS2000-1121.

REFERENCES

AGUILAR, M. J., RAMIREZ DEL POZO, J., AND O. RIBA. 1971. Algunasprecisiones sobre la sedimentacion y paleoecologıa del Cretacico in-ferior en la Zona de Utrillas-Villarroya de los Pinares (Teruel). EstudiosGeologicos, 27:497–512.

ALONSO, J., A. ARILLO, E. BARRON, J. C. CORRAL, J. GRIMALT, J. F.LOPEZ, R. LOPEZ, X. MARTINEZ-DELCLOS, V. ORTUNO, E. PENALVER,AND P. R. TRINCAO. 2000. A new fossil resin with biological inclusionsin lower Cretaceous deposits from Alava (Northern Spain, Basque-Cantabrian Basin). Journal of Paleontology, 74(1):158–178.

ARBIZU, M., E. BERNARDEZ, E. PENALVER, AND M. A. PRIETO. 1999. Elambar de Asturias (Espana). Estudios del Museo de Ciencias Naturalesde Alava, 14(Num. Espec. 2):245–254.

ARILLO, A., AND M. B. MOSTOVSKI. 1999. A new genus of Priophorinae(Diptera, Phoridae) from the Lower Cretaceous amber of Alava (Spain).Studia Dipterologica, 6(2):251–255.

ARILLO, A., AND A. NEL. 2000. Two new fossil cecidomyiids flies fromLower Cretaceous amber of Alava (Spain) (Diptera, Cecidomyiidae).Bulletin de la Societe entomologique de France, 105(3):285–288.

ARILLO, A., AND L. S. SUBIAS. 2000. A new fossil oribatid mite Ar-chaeorchestes minguezae n. gen. n. sp. from the Spanish Lower Cre-taceous amber. Mitteilungen aus dem Geologisch-Palaontogischen. In-stitut der Universitat Hamburg, 84:231–236.

1191ASCASO ET AL.—NEW MICROSCOPE TECHNIQUES FOR AMBER INCLUSIONS

ASCASO, C., J. WIERZCHOS, AND A. DE LOS RIOS. 1998. In situ investi-gations of lichens invading rock at cellular and enzymatic level. Sym-biosis, 24:221–234.

AZAR, D. 1997. A new method for extracting plant and insect fossilsfrom Lebanese amber. Paleontology, 40(4):1027–1029.

AZAR, D., G. FLECK, A. NEL, AND M. SOLIGNAC. 1999a. A new enico-cephalid bug, Enicocephalinus acragrimaldii gen. nov., sp. nov., fromthe Lower Cretaceous amber of Lebanon (insecta, heteroptera, enico-chephalidae). Estudios del Museo de Ciencias Naturales de Alava,14(Num. Espec. 2):217–230.

AZAR, D., A. NEL, M. SOLIGNAC, J.-C. PAICHELER, AND F. BOUCHET.1999b. New genera and species of psychodoid flies from the LowerCretaceous amber of Lebanon. Paleontology, 42(6):1101–1136.

BARRON, E., AND L. ELORZA. 2000. Esporas Muroornati del CretacicoInferior de Penacerrada (Alava, Espana). I Congreso Iberico de Pa-leontologıa/XVI Jornadas de la Sociedad Espanola de Paleontologıa.Evora, 12–14 octubre de 2000, 78–79 p.

BAZ, A., AND V. M. ORTUNO. 2000. Archaeatropidae, a new family ofPsocoptera from the Cretaceous Amber of Alava, Northern Spain. An-nals of Entomologic Society of America, 93(3):367–373.

BAZ, A., AND V. M. ORTUNO. 2001a. A new electrentomoid psocid (Pso-coptera) from the Cretaceous amber of Alava (Northern Spain). Mit-teilungen aus dem Museum fur Naturkunde in Berlin-Deutsch Ento-mologische Zeitschrift 48, 1:27–32.

BAZ, A., AND V. M. ORTUNO. 2001b. New genera and species of em-pheriids (Psocoptera: Empheriidae) from the Cretaceous amber of Al-ava, northern Spain. Cretaceous Research, 22:575–584.

CANN, J. P. 1986. The Feeding Behavior and Structure of Nuclearia de-licatula (Filosea: Aconchulinida). Journal of Protozoology, 33:392–356.

CANO, R. J., AND M. BORUKI. 1995. Revival and identification of bac-terial spores in 25 to 40 million year old Dominican amber. Science,268:1060–1064.

CASAL, G. 1762. Succini Asturici, a Doctore Gafpar Cafal, Almae Ecle-fiae Cathedralis Ovetenfis Medico, reperti, folertique ejufdem cura pro-bati, & examinati, Hiftoria. Historia Natural y Medica del Principadode Asturias. Ed. Facsımil 1988. Servicio de Publicaciones, Oviedo,Principado de Asturias, 480 p.

CORRAL, J. C., R. LOPEZ DEL VALLE, AND J. ALONSO. 1999. El ambarcretacico de Alava (Cuenca Vasco-Cantabrica, Norte de Espana). Sucolecta y preparacion. Estudios del Museo de Ciencias Naturales deAlava, 14(Num spec. 2):7–21.

CRANE, P. R., E. M. FRIIS, AND K. R. PEDERSEN. 1995. The origin andearly diversification of angiosperms. Nature, 374:27–33.

CHERCHI, A., AND R. SCHROEDER. 1982. Sobre la edad de la transgresionmesocretacica en Asturias. Cuadernos de Geologıa Iberica, 8:219–233.

FOISSNER, W. 1991. Basic light and scanning electron microscopic meth-ods for taxonomic studies of ciliated protozoa. European Journal ofProtistology, 27:313–330.

GHIORSE, W. C., AND H. L. EHRLICH. 1992. Microbial mineralization ofiron and manganese. Catena Supplement, 21:75–99.

GRIMALDI, D. A. 1996. Amber, Window to the Past. Harry N. Abrams,Inc., New York, 216 p.

GRIMALDI, D. A., E. BONOWICH, M. DELANNOY, AND S. DOBERSTEIN.1994. Electron microscopic studies of mummified tissues in amber fos-sils. American Museum Novitates, 3097:31 p.

GRIMALDI, D. A., D. AGOSTI, AND J. M. CARPENTER. 1997. New andRediscovered Primitive Ants (Hymenoptera: Formicidae) in CretaceousAmber from New Jersey, and Their Phylogenetic Relationships. Amer-ican Museum Novitates, Number 3208, 43 p.

GRIMALDI, D. A., T. NGUYEN, AND R. KETCHAM. 2000a. Ultra-High-Resolution X-Ray Computed Tomography (UHR CT) and the study offossils in amber, p. 77–91. In D. Grimaldi (ed.), Studies of Fossils inAmber, with Particular Reference to the Cretaceous of New Jersey.Backhuys Publishers, Leiden, The Netherlands.

GRIMALDI, D. A., A. SHEDRINSKY, AND T. W. WAMPLER. 2000b. A re-markable deposit of fossiliferous amber from the Upper Cretaceous(Turonian) of New Jersey, p. 1–77. In D. Grimaldi (ed.), Studies ofFossils in Amber, with Particular Reference to the Cretaceous of NewJersey. Backhuys Publishers, Leiden, The Netherlands.

GRIMALDI, D. A., J. A. LILLEGRAVEN, T. W. WAMPLER, D. BOOKWAL-TER, AND A. SHEDRINSKY. 2000c. Amber from Upper Cretaceousthrough Paleocene strata of the Hanna Basin, Wyoming, with evidencefor source and taphonomy of fossil resins. Rocky Mountain Geology,35:163–204.

HENWOOD, A. 1992a. Exceptional preservation of dipteran flight muscleand the taphonomy of insects in amber. Palaios, 7:203–212.

HENWOOD, A. 1992b. Soft part preservation of beetles in Tertiary amberfrom the Dominican Republic. Paleontology, 35:901–912.

HOFMANN-MUNZ, A. H., H. SCHOPPMANN, AND CH. F. BARDELE. 1990.The oral apparatus of Colpoda variabilis (Ciliophora, Colpodidae), I,3-D reconstruction by serial semi-thin sections and low temperaturescanning electron microscopy. European Journal of Protistology, 26:81–96.

ITURRALDE-VINCENT, M. A., AND R. D. E. MACPHEE. 1996. Age andpaleogeographical origin of Dominican amber. Science, 273:1850–1852.

JOY, D. C. 1991. An introduction to Monte Carlo simulations. ScanningMicroscopy, 5:329–337.

KOHRING, R. 1995. Fossile Bakterien und Pilzsporen aus den BaltischenBernstein. Neues Jahrbuch fur Palaontologie, Monatschefe, 6:321–335.

LAMBERT, L. H., T. COX, K. MITCHELL, R. A. ROSELLO-MORA, C. DEL

CUETO, D. E. DODGE, P. ORKAND, AND R. J. CANO. 1998. Staphylo-coccus succinus sp. nov. isolated from Dominican amber. InternationalJournal of Systematic bacteriology, 48(2):511–518.

LARRASOANA, J. C., AND M. GARCES. 2000. Definicion del contexto delos yacimientos de ambar de Montoria-Penacerrada. Subtarea Paleo-magnetismo. Museo de Ciencias Naturales de Alava. 16 p.

NASCIMBENE, P., AND H. SILVERSTEIN. 2000. The preparation of fragileCretaceous ambers for conservation and study of organismal inclu-sions, p. 93–102. In D. Grimaldi (ed.), Studies of Fossils in Amber,with Particular Reference to the Cretaceous of New Jersey. BackhuysPublishers, Leiden, The Netherlands.

POINAR, JR., G. O. 1992. Life in Amber. Stanford University Press, Stan-ford, California, 350 p.

POINAR, JR., G. O., AND R. HESS. 1982. Ultrastructure of 40-million-year-old insect tissue. Science, 215:1241–1242.

POINAR, JR., G. O., AND R. POINAR. 1999. The Amber Forest. PrincetonUniversity Press, Princeton, New York, 239 p.

POINAR, JR., G. O., E. B. PETERSON, AND J. L. PLATT. 2000. FossilParmelia in New World amber. Lichenologist, 32:263–270.

RAMIREZ DEL POZO, J., AND M. J. AGUILAR. 1969. Ciclotemas en elAptense superior y Albense inferior de Nograro (Alava). Acta Geolo-gica Hispanica, 4:113–118.

RAUTUREAU, M., R. U. COOKE, AND A. BOYDE. 1993. The applicationof confocal microscopy to the study of stone weathering. Earth SurfaceProceedings Landforms, 18:769–775.

RIKKINEN, J., AND G. O. POINAR. 2001. Fossilized fungal mycelium fromTertiary Dominican amber. Mycological Research, 105:890–896.

SCHLEE, D., AND H. G. DIETRICH. 1970. Insektenfuhrender Bernstein ausder Unterkreide des Lebanon. Neues Jahrbuch fur Geologie und Pa-laontologie Monatschefe, 1:40–50.

SCHMIDT, A. R., H. VON EYNATTEN, AND M. WAGREICH. 2001. TheMesozoic amber of Schliersee (southern Germany) is Cretaceous inage. Cretaceous Research, 22:423–428.

SCHONBORN, W., H. DORFELT, W. FOISSNER, L. KRIENITZ, AND U.SCHAFER. 1999. A fossilized microcenosis in Triassic amber. The Jour-nal of Eukaryotic Microbiology, 46:571–584.

SMITH, A. B., AND J. J. AUSTIN. 1997. Can geologically ancient DNAbe recovered from the fossil record? Geoscientist, 7(5):8–11.

STANKIEWICZ, B. A., H. N. POINAR, D. E. G. BRIGGS, R. P. EVERSHED,AND G. O. POINAR JR. 1998. Chemical preservation of plants and in-sects in natural resins. Proceedings of the Royal Society of London B,256:641–647.

SZADZIEWSKI, R., AND A. ARILLO. 1998. Biting midges (Diptera: Cera-topogonidae) from the Lower Cretaceous Amber from Alava, Spain.Polish Journal of Entomology, 67:291–298.

WAGGONER, B. M. 1994. An aquatic microfossil assemblage from Cen-omanian amber of France. Lethaia, 27:77–84.

1192 JOURNAL OF PALEONTOLOGY, V. 77, NO. 6, 2003

WATERS, S. B., AND A. ARILLO. 1999. A new Hybotidae (Diptera, Em-pidoidea) from Lower Cretaceous amber of Alava (Spain). Studia Dip-terologica, 6(1):59–66.

WEITSCHAT, W., AND W. WICHARD. 1998. Atlas der Pflanzen und Tiereim Baltischen Bernstein. Verlag Dr. Friedrich Pfeil, Munchen, 256 p.

WIER, A., M. DOLAN, D. GRIMALDI, R. GUERRERO, J. WAGENSBURG,AND L. MARGULIS. 2002. Spirochete and protist symbionts of a termite(Mastotermes electrodominicus) in Miocene amber. Proceedings of theNational Academy of Sciences, USA, 99:1410–1413.

WIERZCHOS, J., AND C. ASCASO. 1994. Application of backscattered elec-tron imaging to the study of the lichen rock interface. Journal of Mi-croscopy-Oxford, 175:54–59.

WIERZCHOS, J., AND C. ASCASO. 2001. Life, decay and fossilisation ofendolithic microorganisms from the Ross Desert, Antarctica: sugges-tions for in situ further research. Polar Biology, 24:863–868.

WILMSEN, M. 1997. Das Oberalb und Cenoman im NordkantabrischenBecken (Provinz Kantabrien, Nordspanien): Faziesentwicklung, Bio-und Sequenzstratigraphie. Berliner Geowissenschaftliche Abhandlun-gen, (E) 23, 167 p.

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