Combination Of Light Microscopy Research Articles

Morphological and Ultrastructural Features of Selected Epidendroideae Pollen Dispersal Units and New Insights into Their Chemical Nature.

Orchidaceae display enormous diversity in their flower morphology, which is particularly evident in their pollen dispersal units (pollinia, pollinaria). The packaging of pollen by elastoviscin leads to a great diversity of these morphologically and structurally complex pollen units. Despite being one of the most diverse angiosperm families, the available palynological data on orchids remain limited and sometimes contradicting. This study provides new insights into the pollen morphology and ultrastructure of five orchid species from the subfamily Epidendroideae, using combined light, scanning electron, and transmission electron microscopy. The aim was to compare the morphology and ultrastructure of pollen dispersal units and to elucidate the chemical nature of the pollen wall layers and of elastoviscin. Our combined light and electron microscopy investigation demonstrated the presence of six tetrad types even within a single pollinium, which is unique for orchids. The application of different staining methods confirmed the assumed lipidic nature of elastoviscin and the differences in its contrast and ultrastructure suggest a mixture of sticky materials with dissimilar chemical compositions. This study affirmed that sporopollenin is mostly restricted to the outer pollen grains of peripheral tetrads in compact and sectile pollinia, while inner tetrads exhibit highly reduced non-sporopollenin pollen walls.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

Combined light and electron microscopy (CLEM) to quantify methamphetamine-induced alpha-synuclein-related pathology

Methamphetamine (METH) produces a cytopathology, which is rather specific within catecholamine neurons both in vitro and ex vivo, in animal models and chronic METH abusers. This led some authors to postulate a sort of parallelism between METH cytopathology and cell damage in Parkinson’s disease (PD). In fact, METH increases and aggregates alpha-syn proto-fibrils along with producing spreading of alpha-syn. Although alpha-syn is considered to be the major component of aggregates and inclusions developing within diseased catecholamine neurons including classic Lewy body (LB), at present, no study provided a quantitative assessment of this protein in situ, neither following METH nor in LB occurring in PD. Similarly, no study addressed the quantitative comparison between occurrence of alpha-syn and other key proteins and no investigation measured the protein compared with non-protein structure within catecholamine cytopathology. Therefore, the present study addresses these issues using an oversimplified model consisting of a catecholamine cell line where the novel approach of combined light and electron microscopy (CLEM) was used measuring the amount of alpha-syn, which is lower compared with p62 or poly-ubiquitin within pathological cell domains. The scenario provided by electron microscopy reveals unexpected findings, which are similar to those recently described in the pathology of PD featuring packing of autophagosome-like vesicles and key proteins shuttling autophagy substrates. Remarkably, small seed-like areas, densely packed with p62 molecules attached to poly-ubiquitin within wide vesicular domains occurred. The present data shed new light about quantitative morphometry of catecholamine cell damage in PD and within the addicted brain.

Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage

In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.

TEM and EDS characterization in a Bennettitalean cuticle from the Lower Cretaceous Springhill Formation, Argentina

New cuticle samples from the bennettitalean Ptilophyllum eminelidarum were herein studied using the combination of light microscopy (LM), scanning and transmission electron microscopy (SEM, TEM), and element analysis by Energy Dispersive Spectroscopy (EDS). The new TEM studies correspond with and improve the former ones; however, statistical measurements reinforce their uniqueness and erect Bennettitales as a heterogeneous group of cuticles. Six types of cuticles were recognized. According to the factorial component analysis (FCA), the cuticles of the stomatal apparatus show the highest level of interest, while the epidermal cell cuticles, the lowest. For the 9 variables, the top 6 as to importance were the most independent, which could be used as criteria for identification in Bennettitales. A dichotomous key was built, allowing for a rapid identification of each of the 6 types of cuticles based on the 9 variables. Showcasing an upper cuticle significantly thicker and more exposed, and a thinner lower cuticle that is normally more protected, the micropalaeoenvironment of these leaves is of interest. The taxonomical identity of the present taxon is also revealed by the EDS values; S/Ca ratio is homogeneous for all layers of both cuticles, leading to assume, with extreme precaution, a putative similar influence of the palaeoenvironment on these two opposite surfaces of the leaf. As the first detailed statistical TEM and EDS for the Bennettitales, it highlights the great interest of this type of studies and the strong interest for revealing the characters of each type of cuticle and layer.

A rapid workflow for neuron counting in combined light sheet microscopy and magnetic resonance histology.

Information on regional variation in cell numbers and densities in the CNS provides critical insight into structure, function, and the progression of CNS diseases. However, variability can be real or a consequence of methods that do not account for technical biases, including morphologic deformations, errors in the application of cell type labels and boundaries of regions, errors of counting rules and sampling sites. We address these issues in a mouse model by introducing a workflow that consists of the following steps: 1. Magnetic resonance histology (MRH) to establish the size, shape, and regional morphology of the mouse brain in situ. 2. Light-sheet microscopy (LSM) to selectively label neurons or other cells in the entire brain without sectioning artifacts. 3. Register LSM volumes to MRH volumes to correct for dissection errors and both global and regional deformations. 4. Implement stereological protocols for automated sampling and counting of cells in 3D LSM volumes. This workflow can analyze the cell densities of one brain region in less than 1 min and is highly replicable in cortical and subcortical gray matter regions and structures throughout the brain. This method demonstrates the advantage of not requiring an extensive amount of training data, achieving a F1 score of approximately 0.9 with just 20 training nuclei. We report deformation-corrected neuron (NeuN) counts and neuronal density in 13 representative regions in 5 C57BL/6J cases and 2 BXD strains. The data represent the variability among specimens for the same brain region and across regions within the specimen. Neuronal densities estimated with our workflow are within the range of values in previous classical stereological studies. We demonstrate the application of our workflow to a mouse model of aging. This workflow improves the accuracy of neuron counting and the assessment of neuronal density on a region-by-region basis, with broad applications for studies of how genetics, environment, and development across the lifespan impact cell numbers in the CNS.

Pollen characteristics used in determination and systematics of Quercus (Fagaceae): new data and verification of previous concepts

Abstract Quercus (oaks) is a large and important genus of woody angiosperms occurring in a wide range of environments and often occupying a dominant position in temperate forests of the Northern Hemisphere or being a minor component in some subtropical and tropical forests. A reliable determination of fossil dispersed oak pollen requires combined light, scanning electron (SEM) and transmission electron microscopy (TEM) studies and relevant comparative data on extant oak species. We provide SEM and TEM data on 22 extant Quercus spp., representing all sections of the genus. The main objectives of our study were to clarify the nature of the so-called geniculus, a characteristic equatorial bulge in the exine of the colpus found in many oak species, and to test previous hypotheses about the structural basic units of the exine in Quercus. Our ontogenetic study and oxidative experiment involving several Quercus spp. and one Trigonobalanus sp. resulted in a more accurate interpretation of the nature of the geniculus and exine elements, their different developmental states and, therefore, their more reliable application in the systematics and evolution of the genus Quercus and Fagaceae in general.

Disentangling cryptic species in Parastenocarididae (Copepoda: Harpacticoida) with an integrative approach: the case of Stammericaris similior sp. nov. and Stammericaris destillans Bruno & Cottarelli 2017.

Stammericaris similior sp. nov. is described combining light microscopy, scanning electron microscopy, and genetic barcoding. The new species was collected from rimstone pools in Scrivilleri Cave, a cave in Sicily with so far unexplored microcrustacean fauna. The new species is particularly interesting because it is morphologically very similar to Stammericaris destillans, an epikarstic parastenocaridid endemic to a different Sicilian cave; however, the phylogenetic analysis based on the mitochondrial COI gene of sixteen parastenocaridids shows that these two Stammericaris are two distinct species, with an uncorrected p-distance of 22.9, and the sequences of Stammericaris similior sp. nov. cluster together in a well-supported monophyletic clade, with two different haplotypes. To our knowledge, the presence of different species of almost identical morphology had not been recorded before for the genus Stammericaris. The integrated molecular and morphological analysis, the latter conducted with the support of SEM, allows disentangling the affinities of the new species and identifying a few distinctive characters: the males of the new species are characterized by the caudal rami shorter than the anal somite; the morphology of the P3, which is thin and slightly arched, with three proximal spinules on exp-1; the peculiar structure of the P4 enp; the P4 basis ornamented with two spinules of different length, the one closest to the endopod being the shortest one, and a half-moon shaped lamella. The new species differs from S. destillans for its larger size, the presence of: three spinules, instead of two, on the P3 exp-1; the half-moon shaped lamella on the P4 basis; a row of spinules along the inner margin of P4 exp-1. We also provide data on the ecology and distribution of the new species, a list of the other copepod species collected, and a dichotomic key for the males of all species presently assigned to the genus.

The effects of Jurassic igneous intrusions on the generation and retention of gas shale in the Lower Permian source-reservoir shales of Karoo Basin, South Africa

Shale gas potential of the Whitehill Formation (Permian), Karoo Basin, South Africa is strongly influenced by widespread Jurassic igneous intrusions. A high-resolution organic petrographic and geochemical study is reported here on two stratigraphic sections, the objective being to gain insights into the chemical structure and alteration of oil-prone sedimentary organic matter during contact metamorphism. One section unaffected by intrusion served as background, while the other section was intruded by two laterally continuous dolerite sills with an average thickness of 10.7 and 9.3 m, preferentially emplaced at the upper and lower contacts of the formation, respectively. Heat flow from the sills caused TOC and S2 values from programmed pyrolysis to decline over a distance of 8.7 m from 3.2 to 0.15% and 4.65 to 0.11 mg HC/g TOC, respectively, and cause the reflectance of vitrinite and solid bitumen to increase from 2.03 to 5.82% and 1.58 to 7.87%.Organic matter in the background samples is dominated by solid bitumen, a secondary thermal conversion product of oil-generative Type II kerogen. In the sill-hosting section, the thermal aureoles can be recognized by systematic changes to the optical texture and chemical structure and composition of solid bitumen. Solid bitumen particles develop anisotropy approaching the sills and become transformed into coke and pyrolytic carbon. δ13Corg values become less negative by up to 3.13‰ VPDB. These data suggest significant devolatilization of organic matter to methane and destruction of petroleum generation and retention potential in the contact aureole of the sills. The trend of these datasets, coupled with the presence of several sills and their laterally extensive nature throughout much of the study area suggests that the intrusions have been significant in increasing methane generation on a basinal scale. Together, this study indicates that the complementary application of combined light and electron microscopy (CLEM), programmed pyrolysis, and isotope ratio mass spectrometry (IRMS) is suitable for a comprehensive characterization of organic matter alteration through the maturation continuum. Results also show that contact metamorphic alteration of organic matter by intrusions produced structural and compositional changes that are distinctive from those expected from geological burial maturation where graphite is the end-product.

A rare case of monoclonal immunoglobulin deposition disease identified on renal biopsy

Monoclonal light chain immunoglobulin deposition disease (MIDD) is a rare disease disorders in which monoclonal light chains are deposited within the basement membranes of renal structures, including glomeruli, tubules and vessel walls.1 The vast majority of patients diagnosed with MIDD will have renal insufficiency (80%) and half of MIDD patients have myeloma.2 We present the case of a 52-year-old female with a subacute history of impaired renal function and recent creatinine of 220. A renal biopsy performed at Flinders Medical Centre showed 50% renal cortical scarring with prominent tubular atrophy and peritubular basement membrane thickening on light microscopy. Regions containing periglomerular eosinophilic material were seen that labelled with kappa, favouring periglomerular light chain deposition. Direct immunofluorescence revealed linear labelling with kappa along the tubular basement membranes and electron microscopy showed punctate electron-dense deposits in areas, including the glomeruli and tubular basement membranes. The combined light microscopy, immunofluorescence, and electron microscopy results confirmed the diagnosis of MIDD. A discussion of MIDD is presented.

Coccolithophore assemblage changes over the past 9 kyrs BP from a climate hotspot in Tasmania, southeast Australia

Predicting phytoplankton responses to a changing climate on Tasmania's east coast is presently based on short-term plankton data sets (∼75 years). However, to better predict future phytoplankton composition, it is crucial to understand longer-term variations from geological records. A particularly important group are the coccolithophores given their vital contribution to the oceanic carbon pump.Here, we expand the archive of coccolithophores in southeast Australian waters by analysing coccolith assemblages in a 268 cm-long marine sediment core collected off Maria Island, Tasmania, using light and scanning electron microscopy in combination with sedimentary ancient DNA (sedaDNA) techniques.Coccoliths underwent a shift from a cold to warm-water adapted assemblage at ∼8.2 kyrs BP expressed by a transition in species dominance from the cold-water species Gephyrocapsa muellerae to the warmer-water species Emiliania huxleyi Type A. A period of coccolithophore community instability characterised by reduced diversity and species richness was also detected spanning ∼900 yrs. between 6 and 5 kyrs BP. The latter may be associated with a Mid-Holocene warm period in the Southern Ocean as well as sea-level rise changing the study site from a shallow coastal to deep water habitat.Emiliania huxleyi coccoliths displayed the highest total relative abundance, but less prevalent larger taxa (Calcidiscus, Coccolithus, Helicosphaera) accounted for >50% of total estimated coccolith CaCO3 sequestration, indicating that relatively scarce, densely calcified species do the ‘heavy-lifting’ of this process. Analysis of sedaDNA revealed coccolithophores contributed ∼44% to the total palaeo eukaryote composition, underlining their importance as part of the marine ecosystem in the study region. The detection of oceanographic shifts and subsequent coccolithophore assemblage composition, including past transitions of species dominance, offer valuable insight into the biological future of southeast Australian waters.

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM).

Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

Single-Cell Level Measurements of Photocatalytic Damage to Escherichia coli Cells

The increase in water demand due to the increasing population and the addition of emerging contaminants like drug-resistant bacteria, pharmaceuticals, and treatment-resistant chemicals such as PFAS are straining the current water treatment infrastructure. This straining infrastructure could be complemented by photocatalysis and other Advanced oxidation processes (AOP) [1] [2]. Photocatalytic water treatment methods, specifically, have generated a lot of interest due to their ability in eliminating pollutants like dyes and other toxic organic pollutants as well as eliminating pathogens such as bacteria, fungi, and viruses while minimizing the use of material resources. The elimination of bacteria by photocatalysis has been reported widely, with many reports focusing on the kinetics of photocatalytic disinfection, as well as some aspects of process scale-up. However, the mechanism for photocatalytic damage to the bacterial cells has thus far proven difficult to study [3] [4].It is critical to determine the mechanisms of photocatalytic disinfection for the development of better methods of catalyst discovery and reactor design. The mechanisms can be determined if quantitative data on how single bacterial cells interact with catalysts is available. However, experimental measurements at a single-cell level are relatively few and assays for evaluating photocatalyst performance in real-time have not been developed to our knowledge. In this work, we addressed these shortcomings with a combination of light microscopy and particle-tracking algorithms. Using nanowire-shaped titanium dioxide (TiO2) catalyst particles, which maintain a high surface area while remaining visible using the established techniques for light microscopy, we investigated the interactions between catalyst particles and bacteria. We quantified cell motility during the course of photocatalytic disinfection as a loss in motility is expected to faithfully track damage to the membrane [5]. We further correlated the kinetics of antibacterial action of the photocatalytic treatment in E. coli with real-time changes in membrane integrity. Our investigations are anticipated to reveal the mechanisms by which photocatalytic treatments cause cell damage. Such single-cell approaches are expected to help build libraries of photocatalysts and optimize the use of antibacterial agents against superbugs, with a range of future applications in water-disinfection reactors to hospitals.REFERENCES[1] X. Qu, J. Brame, Q. Li, P.J. Alvarez, Nanotechnology for a safe and sustainable water supply: enabling integrated water treatment and reuse, Accounts of chemical research, 46 (2013) 834-843.[2] A. Boretti, L. Rosa, Reassessing the projections of the world water development report, NPJ Clean Water, 2 (2019) 1-6.[3] O.K. Dalrymple, E. Stefanakos, M.A. Trotz, D.Y. Goswami, A review of the mechanisms asnd modeling of photocatalytic disinfection, Applied Catalysis B: Environmental, 98 (2010) 27-38.[4] H.A. Foster, I.B. Ditta, S. Varghese, A. Steele, Photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity, Applied microbiology and biotechnology, 90 (2011) 1847-1868.[5] R. Gupta, K. Y. Rhee, S. D. Beagle, R. Chawla, N. Perdomo, S. W. Lockless and P. P. Lele, Indole modulates cooperative protein-protein interactions in the flagellar motor, PNAS Nexus (2022)

Detaling morphological traits of Trollius europeus L. flowers, nectary structure, and holocrine nectar secretion through combined light and electron microscopy

Trollius europeus L. flowers produce nectar available to various groups of insects. No anatomical studies of these floral nectaries have been conducted to date. This study presents the structure of nectaries at different levels of organisation and the characteristics of petaloids, nectary leaves (petals), and stamens, including their micromorphology. The analyses of the nectaries were carried out with the use of light, fluorescence, scanning electron, and transmission electron microscopy techniques. The nectary located in a small cavity on the nectary leaf consists of the epidermis, nectar-producing parenchyma, and subnectary parenchyma, which has three large vascular bundles containing phloem and xylem. Amyloplasts with starch granules are present only in the parenchyma surrounding the vascular bundles. The other cells of the nectary parenchyma contain only chromoplasts with large plastoglobules. Since there are no chloroplasts, sugars required for nectar production are assumed to originate from phloem sap. The numerous plasmodesmata in the cell walls indicate a symplastic route of pre-nectar. Nectar is secreted onto the nectary surface in the holocrine mode. After disorganisation of the cytoplasmic structure, the epidermal cell wall is disrupted and the cell contents along with the nectar are released into the depression on the nectary leaf. The secretion of nectar from cells is non-synchronous and lasts 4–5 days. The content of nectar proteins and lipids derived from the cytoplasm of epidermal cells increases the nutritional value of the secretion, which may be important for pollinators.

Developmental Plasticity of the Amphibious Liverwort Riccia fluitans.

The colonization of land by ancestors of embryophyte plants was one of the most significant evolutionary events in the history of life on earth. The lack of a buffering aquatic environment necessitated adaptations for coping with novel abiotic challenges, particularly high light intensities and desiccation as well as the formation of novel anchoring structures. Bryophytes mark the transition from freshwater to terrestrial habitats and form adaptive features such as rhizoids for soil contact and water uptake, devices for gas exchange along with protective and repellent surface layers. The amphibious liverwort Riccia fluitans can grow as a land form (LF) or water form (WF) and was employed to analyze these critical traits in two different habitats. A combination of light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies was conducted to characterize and compare WF and LF morphologies. A complete phenotypic adaptation of a WF plant to a terrestrial habitat is accomplished within 15 days after the transition. Stable transgenic R. fluitans lines expressing GFP-TUBULIN and mCherry proteins were generated to study cell division and differentiation processes and revealed a higher cell division activity in enlarged meristematic regions at LF apical notches. Morphological studies demonstrated that the R. fluitans WF initiates air pore formation. However, these pores are arrested at an early four cell stage and do not develop further into open pores that could mediate gas exchange. Similarly, also arrested rhizoid initial cells are formed in the WF, which exhibit a distinctive morphology compared to other ventral epidermal cells. Furthermore, we detected that the LF thallus has a reduced surface permeability compared to the WF, likely mediated by formation of thicker LF cell walls and a distinct cuticle compared to the WF. Our R. fluitans developmental plasticity studies can serve as a basis to further investigate in a single genotype the molecular mechanisms of adaptations essential for plants during the conquest of land.

New Thai giant pill-millipede species, with new genetic barcoding data (Diplopoda, Sphaerotheriida, Zephroniidae).

Three new species of giant pill-millipedes, Sphaerobelum meridionalis Bhansali Wesener sp. nov., Zephronia chrysomallos Bhansali Wesener sp. nov. and Zephronia erawani Bhansali Wesener sp. nov. are described based on museum samples from Thailand. All three species are described in an integrative manner, combining light microscopy, scanning electron microscopy, CT scans and genetic barcoding. Genetic barcoding was successfully conducted for all holotypes of the new species. In addition, genetic barcoding data of four recently described Thai Zephronia species, Zephronia lannaensis Likhitrakarn Golovatch, 2021 in Likhitrakarn et al. 2021, Z. phrain Likhitrakarn Golovatch, 2021, Z. panhai Srisonchai et al. 2021 and Z. golovatchi Srisonchai et al. 2021, together with new locality records, were added to the dataset. Our dataset includes all published sequences of the family Zephroniidae, including all but one (Z. enghoffi Srisonchai et al., 2021) of the described species from Thailand, Laos and Cambodia. All new species are genetically distant from other Zephroniidae from Thailand and surrounding countries showing uncorrected p-distances of >10 %. S. meridionalis sp. nov. is genetically and morphologically close to a recently described aberrant Sphaerobelum, S. aesculus Rosenmejer Wesener, 2021, as well as an unspecified specimen from Malaysia, and might represent a genus different from Sphaerobelum Verhoeff, 1924. Both new Zephronia species are geographically, morphologically and genetically close to Z. panhai, but differ from the latter by >10% p-distance in the COI gene and numerous morphological characters. Virtual cybertypes of the holotype of Zephronia erawani sp. nov. and of a paratype of Z. chrysomallos sp. nov. were created and made publicly accessible.

Desiccation of the leaf mesophyll and its implications for CO2 diffusion and light processing.

Leaves balance CO2 and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress‐induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non‐stressed and drought conditions using a combination of light microscopy, X‐ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low‐mesophyll porosity that were associated with greater gas‐phase diffusion (g IAS), stomatal and mesophyll (g m) conductances and carboxylation capacity. More and highly‐packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought‐induced reductions in mesophyll cell volume, yet the associated increases in porosity and g IAS were obscured by declines in biochemical activity that decreased g m. Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought‐induced changes in leaf structure impacted performance via imposing species‐specific limitations to light absorption, gas exchange and hydraulics.

Conulariids from the Silurian (late Telychian) Waukesha Lagerstätte, Wisconsin

ABSTRACT The Silurian (late Telychian) Brandon Bridge Formation, Wisconsin, USA, is renowned for hosting the Waukesha Konservat-Lagerstätte, in which several rare, enigmatic constituents have received detailed taxonomic and taphonomic study. However, more common species from this deposit remain poorly described. This study provides the first systematic description of two conulariid species documented from the Waukesha Konservat-Lagerstätte – Conularia niagarensis and Metaconularia cf. manni – and assesses their general taphonomy and preservation within this deposit. Specimens were examined using a combination of light microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy to assist in taxonomic, taphonomic and compositional analyses. Conulariid specimens display an array of preservational modes, primarily diagenetic phosphatisation and kerogenization, with minor associated pyritisation. This result suggests a taphonomic history involving post-burial demineralisation associated with falling pH conditions, subsequent compression with minimal breakage, and finally diagenetic mineralisation of the compressed thecae by francolite.

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy.

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

High-resolution Spatiotemporal Dynamics of Harmful Algae in the Indian River Lagoon (Florida)-A Case Study of Aureoumbra lagunensis, Pyrodinium bahamense, and Pseudo-nitzschia.

The Indian River Lagoon (IRL), located on the east coast of Florida, is a complex estuarine ecosystem that is negatively affected by recurring harmful algal blooms (HABs) from distinct taxonomic/functional groups. Enhanced monitoring was established to facilitate rapid quantification of three recurrent bloom taxa, Aureoumbra lagunensis, Pyrodinium bahamense, and Pseudo-nitzschia spp., and included corroborating techniques to improve the identification of small-celled nanoplankton (<10 μm in diameter). Identification and enumeration of these target taxa were conducted during 2015-2020 using a combination of light microscopy and species-specific approaches, specifically immunofluorescence flow cytometry as well as a newly developed qPCR assay for A. lagunensis presented here for the first time. An annual bloom index (ABI) was established for each taxon based on occurrence and abundance data. Blooms of A. lagunensis (>2×108 cells L-1) were observed in all six years sampled and across multiple seasons. In contrast, abundance of P. bahamense, largely driven by the annual temperature cycle that moderates life cycle transitions and growth, displayed a strong seasonal pattern with blooms (105-107 cells L-1) generally developing in early summer and subsiding in autumn. However, P. bahamense bloom development was delayed and abundance was significantly lower in years and locations with sustained A. lagunensis blooms. Pseudo-nitzschia spp. were broadly distributed with sporadic bloom concentrations (reaching 107 cells L-1), but with minimal concentrations of the toxin domoic acid detected (<0.02 μg L-1). In summer 2020, multiple monitoring tools characterized a novel nano-cyanobacterium bloom (reaching 109 cells L-1) that coincided with a decline in A. lagunensis and persisted into autumn. Statistical and time-series analyses of this spatiotemporally intensive dataset highlight prominent patterns in variability for some taxa, but also identifies challenges of characterizing mechanisms underlying more episodic yet persistent events. Nevertheless, the intersect of temperature and salinity as environmental proxies proved to be informative in delineating niche partitioning, not only in the case of taxa with long-standing data sets but also for seemingly unprecedented blooms of novel nanoplanktonic taxa.