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Utilization of integrated correlative light and electron microscopy (iCLEM) for imaging sedimentary organic matter

We report here a new microscopic technique for imaging and identifying sedimentary organic matter in geologic materials that combines inverted fluorescence microscopy with scanning electron microscopy and allows for sequential imaging of the same region of interest without transferring the sample between instruments. This integrated correlative light and electron microscopy technique is demonstrated with observations from an immature lacustrine oil shale from the Eocene Green River Mahogany Zone and mid‐oil window paralic shale from the Upper Cretaceous Tuscaloosa Group. This technique has the potential to allow for identification and characterization of organic matter in shale hydrocarbon reservoirs that is not possible using either light or electron microscopy alone, and may be applied to understanding the organic matter type and thermal regime in which organic nanoporosity forms, thereby reducing uncertainty in the estimation of undiscovered hydrocarbon resources.

Journal of Microscopy

Finalization of the Confocal Laser Scanning Microscopy (CLSM) working group

A working group in Commission II to investigate applications of confocal laser scanning microscopy (CLSM) for organic petrology investigations has finalized with publication of the manuscript “Characterization of bituminite in Kimmeridge Clay by confocal laser scanning and atomic force microscopy” in the International Journal of Coal Geology. The manuscript is available via Open Access from https://doi.org/10.1016/j.coal.2022.103927 and also from the Commission II working group (WG) webpage https://www.iccop.org/workinggroup/confocal-laser-scanning-microscopy-clsm/. A report detailing the full history and results from the WG also is available from the Commission II WG webpage. The working group investigated the application of CLSM to an organic-rich (44 wt.% TOC), thermally immature sample (VRo 0.42%) of the Kimmeridge Clay Formation. CLSM imaging and spectroscopy and atomic force microscopy (AFM) were used to characterize bituminite.

ICCP News

Gross and microscopic morphology of lesions in Cnidaria from Palmyra Atoll, Central Pacific

We conducted gross and microscopic characterizations of lesions in Cnidaria from Palmyra Atoll, Central Pacific. We found growth anomalies (GA) to be the most commonly encountered lesion. Cases of discoloration and tissue loss were rare. GAs had a focal or multi-focal distribution and were predominantly nodular, exophytic, and umbonate. In scleractinians, the majority of GAs manifested as hyperplasia of the basal body wall (52% of cases), with an associated absence or reduction of polyp structure (mesenteries and filaments, actinopharynx and tentacles), and depletion of zooxanthellae in the gastrodermis of the upper body wall. In the soft corals Sinularia sp. and Lobophytum sp., GAs exclusively manifested as prominent hyperplasia of the coenenchyme with an increased density of solenia. In contrast to scleractinians, soft coral GAs displayed an inflammatory and necrotizing component with marked edema of the mesoglea, accompanied by infiltrates of variably-sized granular amoebocytes. Fungi, algae, sponges, and Crustacea were present in some scleractinian GAs, but absent in soft coral GAs. Fragmentation of tissues was a common finding in Acropora acuminata and Montipora cf. dilatata colonies with tissue loss, although no obvious causative agents were seen. Discoloration in the zoanthid, Palythoa tuberculosa, was found to be the result of necrosis, while in Lobophytum sp. discoloration was the result of zooxanthellar depletion (bleaching). Soft corals with discoloration or tissue loss showed a marked inflammatory response, however no obvious causative organisms were seen. Lesions that appeared similar at the gross level were revealed to be distinct by microscopy, emphasizing the importance of histopathology.

Journal of Invertebrate Pathology

The effect of brine on the electrical properties of methane hydrate

Gas hydrates possess lower electrical conductivity (inverse of resistivity) than either seawater or ice, but higher than clastic silts and sands, such that electromagnetic methods can be employed to help identify their natural formation in marine and permafrost environments. Controlled laboratory studies offer a means to isolate and quantify the effects of changing individual components within gas‐hydrate‐bearing systems, in turn yielding insight into the behavior of natural systems. Here we investigate the electrical properties of polycrystalline methane hydrate with ≥25% gas‐filled porosity and in mixture with brine. Initially, pure methane hydrate was synthesized from H 2 O ice and CH 4 gas while undergoing electrical impedance measurement, then partially dissociated to assess the effects of pure pore water accumulation on electrical conductivity. Methane hydrate + brine mixtures were then formed by either adding NaCl (0.25–2.5 wt %) to high‐purity ice or by using frozen seawater as a reactant. Conductivity was obtained from impedance measurements made in situ throughout synthesis while temperature cycled between +15 °C and −25 °C. Several possible conduction mechanisms were subsequently determined using equivalent circuit modeling. Samples with low NaCl concentration show a doping/impurity effect and a log linear conductivity response as a function of temperature. For higher salt content samples, conductivity increases exponentially with temperature and the log linear relationship no longer holds; instead, we observe phase changes within the samples that follow NaCl–H 2 O–CH 4 phase equilibrium predictions. Final samples were quenched in liquid nitrogen and imaged by cryogenic scanning electron microscopy (cryo‐SEM) to assess grain‐scale characteristics.

Journal of Geophysical Research

Plasma chloride and gill epithelial response of channel catfish to infection with Ichthyophthirius multifillis

Because infection with Ichthyophthirius multifiliis has been reported to result in osmotic stress, genesis of the plasma chloride and chloride cell response of channel catfish Ictalurus punctatus to the infection was studied from day 0 to day 7 postexposure (PE). Parasite population densities ranged from 0.08 to 0.22 trophonts per gill filament during days 1–5 PE and increased to 2.1 at day 7 PE. Population increases were the result of infection by a new generation of infective theronts and reproduction by the parasite on the host. Mean plasma chloride concentrations of fish were not significantly different among days 0–5 PE (range of means, 93.01–97.87 meq/L), but by day 7 PE the mean had declined significantly, to 83.73 meq/L. Maintenance of relatively stable plasma chloride concentrations for 5 d was associated with chloride cell proliferation. The number of interlamellar filament chloride cells had significantly increased by day 1 PE, and the number of chloride cells in the lamellae had significantly increased by day 5 PE. No further increase had occurred by day 7 PE. Vacuolization of chloride cells, examined by electron microscopy, became more common as infection progressed, and many chloride cells appeared to be degenerating by day 5 PE.

Journal of Aquatic Animal Health

Pathology of tissue loss in three key gorgonian species in the Mediterranean Sea

The Mediterranean is known for its marine biodiversity, especially gorgonian forests. Unfortunately, these are experiencing rapid declines due to climate change, manifested by repeated marine heat waves resulting in mass mortality events since the early 1990 s. To better understand why gorgonians are declining, more systematic approaches to investigate the exact causes are needed, and pathology may aid in this goal. We described gross and microscopic pathology of tissue loss in three key gorgonian species in the Mediterranean region, Paramuricea clavata , Eunicella cavolini , and Leptogorgia sarmentosa , that were all experiencing various degrees of acute to subacute tissue loss characterized by exposed axial skeleton sometimes partly colonized by epibionts and thinning of adjacent tissues. The most significant variety of lesions was seen in P. clavata followed by L. sarmentosa and E. cavolini. For all species, dissociation of gastrodermal cells was the dominant microscopic lesion followed by necrosis of the gastrodermis. Ciliates invading gastrodermis and associated with necrosis of polyps were seen only in E. cavolini. Epidermal tissue loss was seen only in L. sarmentosa , while P. clavata was distinguished by a prominent inflammatory response and unidentified dark round structures within the tentacle epidermis and gastrodermis with no host response. Further work to understand the cause of death in gorgonians is needed, particularly to elucidate the role of ciliates and environmental co-factors or infectious agents not visible on light microscopy, as well as applications of additional tools such as cytology.

Journal of Invertebrate Pathology

Recognition of oxidized sulfide minerals as an exploration guide for uranium

The difference in color between tan to red oxidized sandstone and gray reduced sandstone on either side of the reduction-oxidation (redox) interface, which is- the locus of uranium concentration in roll-type deposits, has been noted and used locally as an exploration guide within known uranium districts. Reduced sandstone is characterized in many deposits by the presence of iron sulflde minerals (particularly marcasite and pyrite) that occur as replacements of and overgrowths on iron-titanium oxide minerals and plant debris and as cement of detrital grains. Oxidation of the sulfldes by oxygenated ground water forms yellow to red ferric oxide and ferric hydroxide phases ("limonite") that replace the FeS 2 minerals. Processes other than the oxidation of sulfldes, however, can cause reddening of clastic sediments without the creation of a redox boundary, and so color alone is not a sufficient condition by which to judge the potential for uranium ore in frontier areas. Ferric oxides that form by the oxidation of iron sulflde minerals can be distinguished from other forms of ferric oxide by reflection microscopic examination of polished grain mounts- and polished thin sections'. Diagnostic features of oxidized sulfldes are limonite pseudomorphs of sulfldes and limonite containing internal textures that mimic characteristic textures of previously sulfldized detrital phases. Recognition of oxidized sulfides by reflection microscopy, then, can distinguish oxidized sandstones, which might have a redox boundary downdip and which would therefore be favorable hosts for uranium concentration, from those without such potential.

Journal of Research of the U.S. Geological Survey

Naturally occurring vapor-liquid-solid (VLS) Whisker growth of germanium sulfide

The first naturally occurring terrestrial example of vapor-liquid-solid (VLS) growth has been observed in condensates from gases released by burning coal in culm banks. Scanning electron microscopy, X-ray diffraction, and energy dispersive analysis indicate that the crystals consist of elongated rods (≈ 100 μm) of germanium sulfide capped by bulbs depleted in germanium.

Journal of Crystal Growth

Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix , Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.

Journal of Fish Biology

Hemorrhagic enteritis in captive American kestrels (Falco sparverius)

Hemorrhagic enteritis and hepatitis of suspected adenovirus etiology were the apparent cause of death of nine captive American kestrels. Cloacal hemorrhage was the only prominent gross lesion: disseminated hepatocellular necrosis, and intranuclear inclusion bodies were evident microscopically. Electron microscopy revealed numerous adenovirus-like particles associated with the hepatic lesions. Attempts to serologically identify the agent were unsuccessful.

Journal of Wildlife Diseases

Serological responses and immunity to superinfection with avian malaria in experimentally-infected Hawaii Amakihi

Six of seven Hawaii Amakihi ( Hemignathus virens ) with chronic malarial infections had no increases in peripheral parasitemia, declines in food consumption, or loss of body weight when rechallenged with the homologous isolate of Plasmodium relictum 61 to 62 days after initial infection. Five uninfected control amakihi exposed at the same time to infective mosquito bites developed acute infections with high parasitemias. Reductions in food consumption and loss of body weight occurred in all control birds and three of these individuals eventually died. When surviving birds were rechallenged >2 yr later with either the same parasite isolate or an isolate of P. relictum collected on the island of Kauai, all individuals were immune to superinfection. Chronically infected birds developed antibodies to a common suite of malarial antigens ranging in size from 22 to 170 kDa that were detectable as early as 8 days post infection on immunoblots of SDS-polyacrylamide gels. Antibodies to this suite of malarial antigens persisted as long as 1,248 days after initial infection and were consistently detectable at times when parasites were not easily found by microscopy on Giemsa-stained blood smears. The immunoblotting method that is described here appears to be an effective technique for identifying birds with chronic, low-intensity malarial infections when circulating parasites are not easily detectable by microscopy. Hawaiian honeycreepers that are capable of recovering from acute infections develop concomitant immunity to superinfection, making them functionally immune in areas where malaria transmission has become endemic.

Journal of Wildlife Diseases

Detection of parvoviruses in wolf feces by electron microscopy

One hundred fifteen wolf (Canis lupus) feces were collected between 1980 and 1984 from northeastern Minnesota and were examined for canine parvovirus by negative contrast electron microscopy. Of these, seven (6%) samples revealed the presence of parvovirus. Some of these viruses were able to grow in cell cultures forming intranuclear inclusion bodies and giant cells.

Journal of Wildlife Diseases

A new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey ( Petromyzon marinus )

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey ( Petromyzon marinus ) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

Journal of Visualized Experiments

Recommended procedures and techniques for the petrographic description of bituminous coals

Modern coal petrology requires rapid and precise description of great numbers of coal core or bench samples in order to acquire the information required to understand and predict vertical and lateral variation of coal quality for correlation with coal-bed thickness, depositional environment, suitability for technological uses, etc. Procedures for coal description vary in accordance with the objectives of the description. To achieve our aim of acquiring the maximum amount of quantitative information within the shortest period of time, we have adopted a combined megascopic-microscopic procedure. Megascopic analysis is used to identify the distinctive lithologies present, and microscopic analysis is required only to describe representative examples of the mixed lithologies observed. This procedure greatly decreases the number of microscopic analyses needed for adequate description of a sample. For quantitative megascopic description of coal microlithotypes, microlithotype assemblages, and lithotypes, we use (V) for vitrite or vitrain, (E) for liptite, (I) for inertite or fusain, (M) for mineral layers or lenses other than iron sulfide, (S) for iron sulfide, and (X 1 ), (X 2 ), etc. for mixed lithologies. Microscopic description is expressed in terms of V representing the vitrinite maceral group, E the exinite group, I the inertinite group, and M mineral components. volume percentages are expressed as subscripts. Thus (V) 20 (V 80 E 10 I 5 M 5 ) 80 indicates a lithotype or assemblage of microlithotypes consisting of 20 vol. % vitrite and 80% of a mixed lithology having a modal maceral composition V 80 E 10 I 5 M 5 . This bulk composition can alternatively be recalculated and described as V 84 E 8 I 4 M 4 . To generate these quantitative data rapidly and accurately, we utilize an automated image analysis system (AIAS). Plots of VEIM data on easily constructed ternary diagrams provide readily comprehended illustrations of the range of modal composition of the lithologic units making up a given coal bed. The use of bulk-specific-gravity determinations is alo recommended for identification and characterization of the distinctive lithologic units. The availability of an AIAS also enhances the capability to acquire textural information. Ranges of size of maceral and mineral grains can be quickly and precisely determined by use of an AIAS. We assume that shape characteristics of coal particles can also be readily evaluated by automated image analysis, although this evaluation has not yet been attempted in our laboratory. Definitive data on the particulate mineral content of coal constitute another important segment of petrographic description. Characterization of mineral content may be accomplished by optical identification, electron microprobe analysis, X-ray diffraction, and scanning and transmission electron microscopy. Individual mineral grains in place in polished blocks or polished this sections, or separated from the coal matrix by sink-float methods are studied by analytical techniques appropriate to the conditions of sampling.

International Journal of Coal Geology

Disseminated granulomas caused by an unidentified protozoan in sandhill cranes

Oral granulomas were observed in 31 (33%) of 95 captive sandhill cranes (Grus canadensis) at the Patuxent Wildlife Research Center. Necropsy of six of the afflicted cranes revealed granulomatous nodules throughout many of their organ systems. Intracellular protozoan organisms morphologically resembling schizogonic stages were observed within the granulomas by light and electron microscopy. Sexual and asexual stages of coccidia were seen in sections of the intestines of 4 of 5 cranes examined microscopically, and Eimerian oocysts were seen in fecal flotation specimens from 3 of 4 birds.

Journal of the American Veterinary Medical Associa

Transglobal spread of an ecologically relevant sea urchin parasite

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

ISME Journal

Mycoplasma testudineum in free-ranging desert tortoises, Gopherus agassizii

We performed clinico-pathological evaluations of 11 wild Agassiz's desert tortoises (Gopherus agassizii) from a translocation project in the central Mojave Desert, California, USA. Group 1 consisted of nine tortoises that were selected primarily due to serologic status, indicating exposure to Mycoplasma testudineum (seven) or both M. agassizii and M. testudineum (two), and secondarily due to clinical signs of upper respiratory tract disease (URTD). Group 2 consisted of two tortoises that were antibody-negative for Mycoplasma and had no clinical signs of URTD, but did have other signs of illness. Of the Group 1 tortoises, M. testudineum, but not M. agassizii, was amplified by polymerase chain reaction and DNA fingerprinted from two tortoises. Using light microscopy, mild to severe pathologic changes were observed in one or more histologic sections of either one or both nasal cavities of each tortoise in Group 1. Our findings support a causal relationship between M. testudineum and URTD in desert tortoises.

California

A review of spatially resolved techniques and applications of organic petrography in shale petroleum systems

This review examines new techniques and applications of organic petrography in source-rock reservoir petroleum systems that have occurred along with development of the global ‘shale revolution’ in energy resources. The review is limited to techniques and instrumentation that provide spatially resolved information, typically at or below microscales, for dispersed organic matter occurring in situ in samples of shale and mudrock. A brief summary of ion beam sample preparation is followed by discussion of the most common analytical techniques and applications. Advantages and limitations of each technique, including requisite sample preparation, types of information generated [e.g., molecular or elemental (isotopic) abundance], sensitivity, and resolution are discussed. In a few cases, techniques not yet applied to organic petrology of shale or mudrock are described (e.g., X-ray photoelectron spectroscopy imaging), in anticipation of near-term future application. The most common in situ techniques applied for characterization of organic matter in shale and mudrock include optical (including fluorescence) and electron microscopies, Raman, fluorescence, and infrared spectroscopies, and surficial measurements via force microscopy. Techniques that show growing application to organic petrography of shale include tip-enhanced photothermal infrared spectroscopy, mass spectrometry imaging, and synchrotron-based spectroscopies, among others. It is anticipated that the future of dispersed organic matter petrography will hold continued development of integrated instrument techniques (e.g., simultaneous or sequential correlative microscopies and/or spectroscopies of the same location), increased instrumental resolution, increased use of multiscale and multimodal organic petrography investigations, and three-dimensional imaging and chemical speciation mapping applications via multiple analytical approaches.

International Journal of Coal Geology