From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur;and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples

Label-free 3D-CLEM Using Endogenous Tissue Landmarks

Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable "flat embedding" preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines

Lack of FIBRILLIN6 in Arabidopsis thaliana affects light acclimation and sulfate metabolism

Arabidopsis thaliana contains 13 fibrillins (FBNs), which are all localized to chloroplasts. FBN1 and FBN2 are involved in photoprotection of photosystem II, and FBN4 and FBN5 are thought to be involved in plastoquinone transport and biosynthesis, respectively. The functions of the other FBNs remain largely unknown. To gain insight into the function of FBN6, we performed coexpression and Western analyses, conducted fluorescence and transmission electron microscopy, stained reactive oxygen species (ROS), measured photosynthetic parameters and glutathione levels, and applied transcriptomics and metabolomics. Using coexpression analyses, FBN6 was identified as a photosynthesis‐associated gene. FBN6 is localized to thylakoid and envelope membranes, and its knockout results in stunted plants. The delayed‐growth phenotype cannot be attributed to altered basic photosynthesis parameters or a reduced CO2 assimilation rate. Under moderate light stress, primary leaves of fbn6 plants begin to bleach and contain enlarged plastoglobules. RNA sequencing and metabolomics analyses point to an alteration in sulfate reduction in fbn6. Indeed, glutathione content is higher in fbn6, which in turn confers cadmium tolerance of fbn6 seedlings. We conclude that loss of FBN6 leads to perturbation of ROS homeostasis. FBN6 enables plants to cope with moderate light stress and affects cadmium tolerance

Two adhesive systems cooperatively regulate axon ensheathment and myelin growth in the CNS

Central nervous system myelin is a multilayered membrane produced by oligodendrocytes to increase neural processing speed and efficiency, but the molecular mechanisms underlying axonal selection and myelin wrapping are unknown. Here, using combined morphological and molecular analyses in mice and zebrafish, we show that adhesion molecules of the paranodal and the internodal segment work synergistically using overlapping functions to regulate axonal interaction and myelin wrapping. In the absence of these adhesive systems, axonal recognition by myelin is impaired with myelin growing on top of previously myelinated fibers, around neuronal cell bodies and above nodes of Ranvier. In addition, myelin wrapping is disturbed with the leading edge moving away from the axon and in between previously formed layers. These data show how two adhesive systems function together to guide axonal ensheathment and myelin wrapping, and provide a mechanistic understanding of how the spatial organization of myelin is achieved

Insights into replicative senescence of human testicular peritubular cells

There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced beta-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target

Validation of a Hypothesis: Colonization of Black Smokers by Hyperthermophilic Microorganisms

Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but house hyperthermophilic microorganisms in substantial amounts in later stages. No direct experimental data exist by which mechanisms hyperthermophiles colonize newly erupted black smokers, but a scenario was proposed recently how this might happen. Here we combine high temperature light microscopy with electron microscopy to show that two hyperthermophilic Archaea, namely Pyrococcus furiosus and Methanocaldococcus villosus are able to adhere onto authentic black smoker material (BSM). We especially are able to directly observe the adhesion process via video recordings taken at high temperatures. These data validate the hypothesis that hyperthermophiles are transferred by serendipitous water currents to the outside of newly formed black smokers and react within seconds to the there prevailing high temperatures by very fast movements. They scan the surface of the hydrothermal chimneys via a much slower zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms

Video3.WMV

<p>Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but house hyperthermophilic microorganisms in substantial amounts in later stages. No direct experimental data exist by which mechanisms hyperthermophiles colonize newly erupted black smokers, but a scenario was proposed recently how this might happen. Here we combine high temperature light microscopy with electron microscopy to show that two hyperthermophilic Archaea, namely Pyrococcus furiosus and Methanocaldococcus villosus are able to adhere onto authentic black smoker material (BSM). We especially are able to directly observe the adhesion process via video recordings taken at high temperatures. These data validate the hypothesis that hyperthermophiles are transferred by serendipitous water currents to the outside of newly formed black smokers and react within seconds to the there prevailing high temperatures by very fast movements. They scan the surface of the hydrothermal chimneys via a much slower zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms.</p

Video2.WMV

<p>Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but house hyperthermophilic microorganisms in substantial amounts in later stages. No direct experimental data exist by which mechanisms hyperthermophiles colonize newly erupted black smokers, but a scenario was proposed recently how this might happen. Here we combine high temperature light microscopy with electron microscopy to show that two hyperthermophilic Archaea, namely Pyrococcus furiosus and Methanocaldococcus villosus are able to adhere onto authentic black smoker material (BSM). We especially are able to directly observe the adhesion process via video recordings taken at high temperatures. These data validate the hypothesis that hyperthermophiles are transferred by serendipitous water currents to the outside of newly formed black smokers and react within seconds to the there prevailing high temperatures by very fast movements. They scan the surface of the hydrothermal chimneys via a much slower zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms.</p

Video5.WMV

<p>Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but house hyperthermophilic microorganisms in substantial amounts in later stages. No direct experimental data exist by which mechanisms hyperthermophiles colonize newly erupted black smokers, but a scenario was proposed recently how this might happen. Here we combine high temperature light microscopy with electron microscopy to show that two hyperthermophilic Archaea, namely Pyrococcus furiosus and Methanocaldococcus villosus are able to adhere onto authentic black smoker material (BSM). We especially are able to directly observe the adhesion process via video recordings taken at high temperatures. These data validate the hypothesis that hyperthermophiles are transferred by serendipitous water currents to the outside of newly formed black smokers and react within seconds to the there prevailing high temperatures by very fast movements. They scan the surface of the hydrothermal chimneys via a much slower zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms.</p