When the Utility of Micro-Computed Tomography Collides with Insect Sample Preparation: An Entomologist User Guide to Solve Post-Processing Issues and Achieve Optimal 3D Models

Many techniques are used today to study insect morphology, including light and electron microscopy. Most of them require to specifically prepare the sample, precluding its use for further investigation. In contrast, micro-CT allows a sample to be studied in a non-destructive and rapid process, even without specific treatments that might hinder the use of rare and hard-to-find species in nature. We used synchrotron radiation (SR) micro-CT and conventional micro-CT to prepare 3D reconstructions of Diptera, Coleoptera, and Hymenoptera species that had been processed with 4 common preparation procedures: critical-point drying, sputter-coating, resin embedding, and air-drying. Our results showed that it is possible to further utilize insect samples prepared with the aforementioned preparation techniques for the creation of 3D models. Specimens dried at the critical point showed the best results, allowing us to faithfully reconstruct both their external surface and their internal structures, while sputter-coated insects were the most troublesome for the 3D reconstruction procedure. Air-dried specimens were suitable for external morphological analyses, while anatomical investigation of soft internal organs was not possible due to their shrinking and collapsing. The sample included in resin allowed us to reconstruct and appreciate the external cuticle and the internal parts. In this work, we demonstrate that insect samples destined to different analyses can be used for new micro-CT studies, further deepening the possibility of state-of-the-art morphological analyses.

Relationships between Intraocular Pressure, Effective Filtration Area and Morphological Changes in the Trabecular Meshwork of Steroid-Induced Ocular Hypertensive Mouse Eyes

We investigated whether an inverse relationship exists between intraocular pressure (IOP) and effective filtration area (EFA) in the trabecular meshwork (TM) in a steroid-induced ocular hypertensive (SIOH) mouse model and the morphological changes associated with the reduction of EFA. C57BL/6 mice (n = 15 per group) received either 0.1% dexamethasone (DEX) or saline eye drops twice daily for five weeks. IOP was measured weekly. Fluorescent tracers were injected into the anterior chamber to label EFA at the endpoint. Injected eyes were fixed and processed for confocal microscopy. EFA in the TM was analyzed. Light and electron microscopy were performed in high- and low-tracer regions of six eyes per group. The mean IOP was ~4 mm Hg higher in DEX-treated than saline-treated control eyes (p < 0.001) at the endpoint. EFA was reduced in DEX-treated eyes compared to controls (p < 0.01) and negatively correlated with IOP (R2 = 0.38, p = 0.002). Reduced thickness of juxtacanalicular tissue (JCT) and increased abnormal extracellular matrix in the JCT were found to be associated with reduced EFA. Our data confirm the inverse relationship between EFA and IOP, suggesting that morphological changes in the JCT contribute to the reduction of EFA, thus elevating IOP in SIOH mouse eyes.

Biogeochemical Niche of Magnetotactic Cocci Capable of Sequestering Large Polyphosphate Inclusions in the Anoxic Layer of the Lake Pavin Water Column

Magnetotactic bacteria (MTB) are microorganisms thriving mostly at oxic–anoxic boundaries of aquatic habitats. MTB are efficient in biomineralising or sequestering diverse elements intracellularly, which makes them potentially important actors in biogeochemical cycles. Lake Pavin is a unique aqueous system populated by a wide diversity of MTB with two communities harbouring the capability to sequester not only iron under the form of magnetosomes but also phosphorus and magnesium under the form of polyphosphates, or calcium carbonates, respectively. MTB thrive in the water column of Lake Pavin over a few metres along strong redox and chemical gradients representing a series of different microenvironments. In this study, we investigate the relative abundance and the vertical stratification of the diverse populations of MTB in relation to environmental parameters, by using a new method coupling a precise sampling for geochemical analyses, MTB morphotype description, and in situ measurement of the physicochemical parameters. We assess the ultrastructure of MTB as a function of depth using light and electron microscopy. We evidence the biogeochemical niche of magnetotactic cocci, capable of sequestering large PolyP inclusions below the oxic–anoxic transition zone. Our results suggest a tight link between the S and P metabolisms of these bacteria and pave the way to better understand the implication of MTB for the P cycle in stratified environmental conditions.

AUTOIMMUNE CHOLESTATIC LESIONS OF BILIARY DUCTS

The review presents literature data and original findings of light and electron microscopy of pathomorphological changes in the bile ducts in primary sclerosing cholangitis (PSC), immunoglobulin G4 (IgG4)-associated autoimmune sclerosing cholangitis and overlap syndromes: PSC + chronic autoimmune hepatitis (AIH); PSC + primary biliary cirrhosis (PBC).

A modular platform for automated cryo-FIB workflows

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Personal Perspectives on Plant Ribosomal RNA Genes Research: From Precursor-rRNA to Molecular Evolution

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss – from a personal view – the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on “Molecular organization, evolution, and function of ribosomal DNA.”

Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection

Speed and efficiency of data collection and image processing in cryo electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged behind and faster, more reproducible specimen preparation devices are needed. Here we present a new vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that quality of the prepared cryo specimen is characterized prior to electron microscopy data acquisition. Practicality and performance of the device are illustrated by experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo electron tomography and cryo correlated light and electron microscopy.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Triangulopteris lacunata gen. et sp. nov. (Centroplasthelida), a New Centrohelid Heliozoan from Soil †

A new genus and species of centrohelid heliozoans, Triangulopteris lacunata gen. et sp. nov. (Pterocystidae Cavalier-Smith and Heyden, 2007), from four geographically remote locations (the Crimean Peninsula, the Dnieper Lowland (the East European Plain), Franz Josef Land, and the Kolyma Lowland (North–Eastern Siberia) was examined using light and electron microscopy. The novel centrohelid is characterized by round shape, 4.3–16.3 μm in diameter, covered with two types of scales: 1.06–4.54 μm long triangular spine scales and 1.22–2.05 μm oval plate scales. Studied centrohelid heliozoan possesses a unique spine scale morphology. The base of scales is represented by a horse hoof-shaped basal plate. The inner surface and lateral wings of spine scales have numerous radial ribs with two ‘pockets’ that are located on both sides of the spine shaft. These pockets are formed by the lateral wings and ends of the basal plate. The cyst formation and transition to a spicules-bearing stage were noted. Additionally, phylogenetic tree was constructed based on SSU rRNA sequences including the strain HF-25 from the permafrost of Kolyma Lowland. The resulting phylogeny recovered it within the clade Pterista, while forming a separate sister lineage to H2 clade, which only had included freshwater environmental sequences.