Human cardiac telocytes: 3D imaging by FIB‐SEM tomography

Telocyte (TC) is a newly identified type of cell in the cardiac interstitium ( www.telocytes.com ). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three‐dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB‐SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB‐SEM tomography confirms that they have long, narrow but flattened (ribbon‐like) telopodes, with humps generated by the podoms. FIB‐SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB‐SEM tomography of a human cell type.     

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.     

FIB‐SEM tomography of human skin telocytes and their extracellular vesicles

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non‐epithelial spaces (stroma) of many organs (see www.telocytes.com ). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB‐SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB‐SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon‐like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB‐SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.     

Simulation of the Planetary Interior Differentiation Processes in the Laboratory

A planetary interior is under high-pressure and high-temperature conditions and it has a layered structure. There are two important processes that led to that layered structure, (1) percolation of liquid metal in a solid silicate matrix by planet differentiation, and (2) inner core crystallization by subsequent planet cooling. We conduct high-pressure and high-temperature experiments to simulate both processes in the laboratory. Formation of percolative planetary core depends on the efficiency of melt percolation, which is controlled by the dihedral (wetting) angle. The percolation simulation includes heating the sample at high pressure to a target temperature at which iron-sulfur alloy is molten while the silicate remains solid, and then determining the true dihedral angle to evaluate the style of liquid migration in a crystalline matrix by 3D visualization. The 3D volume rendering is achieved by slicing the recovered sample with a focused ion beam (FIB) and taking SEM image of each slice with a FIB/SEM crossbeam instrument. The second set of experiments is designed to understand the inner core crystallization and element distribution between the liquid outer core and solid inner core by determining the melting temperature and element partitioning at high pressure. The melting experiments are conducted in the multi-anvil apparatus up to 27 GPa and extended to higher pressure in the diamond-anvil cell with laser-heating. We have developed techniques to recover small heated samples by precision FIB milling and obtain high-resolution images of the laser-heated spot that show melting texture at high pressure. By analyzing the chemical compositions of the coexisting liquid and solid phases, we precisely determine the liquidus curve, providing necessary data to understand the inner core crystallization process.     

Three‐dimensional nanocharacterization of porous hydrogel with ion and electron beams

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water‐soluble metabolites through their high water‐content matrix. The ability to image three‐dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell‐biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB‐tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well. Biotechnol. Bioeng. 2013; 110: 318–326. © 2012 Wiley Periodicals, Inc.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture

Focused ion beam (FIB) milling in combination with field emission scanning electron microscopy (FESEM) was applied to investigations of metaphase barley chromosomes, providing new insight into the chromatin packaging in the chromosome interior and 3D distribution of histone variants in the centromeric region. Whole mount chromosomes were sectioned with FIB with thicknesses in the range of 7–20 nm, resulting in up to 2000 sections, which allow high resolution three-dimensional reconstruction. For the first time, it could be shown that the chromosome interior is characterized by a network of interconnected cavities, with openings to the chromosome surface. In combination with immunogold labeling, the centromere-correlated distribution of histone variants (phosphorylated histone H3, CENH3) could be investigated with FIB in three dimensions. Limitations of classical SEM analysis of whole mount chromosomes with back-scattered electrons requiring higher accelerating voltages, e.g. faint and blurred interior signals, could be overcome with FIB milling: from within the chromosome even very small labels in the range of 10 nm could be precisely visualized. This allowed direct quantification of marker molecules in a three-dimensional context. Distribution of DNA in the chromosome interior could be directly analyzed after staining with a DNA-specific platinorganic compound Platinum Blue. Higher resolution visualization of DNA distribution could be performed by preparation of FIB lamellae with the in situ lift-out technique followed by investigation in dark field with a scanning transmission electron detector (STEM) at 30 kV.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.     

G551D CF mice display an abnormal host response and have impaired clearance of Pseudomonas lung disease

Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.     

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.     

High-precision targeting workflow for volume electron microscopy

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.     

Profiling of RNA ribose methylation in Arabidopsis thaliana

Eukaryotic rRNAs and snRNAs are decorated with abundant 2′-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2′-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.     

Molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 μL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses.Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 μL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m(3) in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5-1.0 μm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.     

A novel approach for intracellular 3D immuno-labeling for electron tomography

Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.     

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.