Introduction
For three-dimensional (3D) ultrastructural investigations with electron microscopy (EM), five different techniques are typically used (Fig. 1). Each one of these techniques, however, has significant drawbacks: (i) classical transmission EM (TEM) serial sectioning, which is largely unsuitable for long series, has many drawbacks, such as compression of sections, uneven stretching, folds, knife marks, etc.; (ii) TEM-tomography, although achieving the highest resolution, is limited in section thickness (max. 1 µm); (iii) serial block face sectioning (3View®), a built-in ultramicrotome within a scanning electron microscopy (SEM), is suitable for large volumes but limited in section thickness (20 nm at best) and is hampered by charging; (iv) array tomography, which is non-destructive, but limited in z-resolution just as classical serial sectioning; and (v) focused ion beam (FIB)/SEM-tomography, which currently offers by far the highest resolution along the z axis with a “section thickness” down to 2 nm for long image series (for review see: Peddie & Collinson, Reference Peddie and Collinson2014; Romero-Brey & Bartenschlager, Reference Romero-Brey and Bartenschlager2015; Karreman et al., Reference Karreman, Hyenne, Schwab and Goetz2016; Xu et al., Reference Xu, Hayworth, Lu, Grob, Hassan, García-Cerdán, Niyogi, Nogales, Weinberg and Hess2017). A period of 10 years after FIB/SEM was recognized as a revolutionary tool for 3D-EM in biology, these instruments are widely accepted as expensive, but highly complementary and efficient tools, which are indispensable for ultrastructural studies. However, the analytical potential of the instrument itself, the SEM, is still not fully exploited. Initially, a SEM was simply considered a surface imaging instrument. However, the use of different detectors and the variation of many SEM parameters offer enormous analytical capacities, well beyond surface imaging.
Three different signals can be routinely used for structural and analytical information, as described by Bozzola & Russell (Reference Bozzola and Russell1991). First, the secondary electrons (SE) give the characteristic topography of SEM images. Second, the backscattered electrons (BSE) provide a material contrast and due to their high energy and sub-surface information. Third, X-ray detection can be harnessed to analyze the local atomic composition (Scala et al., Reference Scala, Pasquinelli, Martegani and Laschi1985; Bozzola & Russell, Reference Bozzola and Russell1991; Utke et al., Reference Utke, Moshkalev and Russell2012; Drobne, Reference Drobne2013). These signals can be monitored separately or in combination and are indispensable for SEM and to some extent required for FIB/SEM.
Despite utilizing multiple signals, a correlation of light microscopy (LM) and EM remains challenging. Reference points or fiducial markers are essential to facilitate a correlation, but a universal labeling does not exist, since the requirements vary widely for different experiments. Many years before they were commercially available, we developed slides (=point finder) with stable, solvent resistant coordinates by sintering transfer pictures (decalcomania; Wanner et al., Reference Wanner, Gaβner, Formanek and Greiner1993). Since the labeling was elevated, experiments such as chromosome spreads were impeded. With new laser technology, it was possible to produce engraved coordinate systems with much finer symbols. These slides were suitable for wider range of biological samples, including chromosome spreads, microorganisms, and proliferating cultured cells. Confocal laser scanning microscopy has broadened the spectrum allowing the production of labeled coverslips.
When correlative light and electron microscopy (CLEM) became fashionable, the scientific interest was primarily focused on improving LM resolution, aiming to image the smallest structures first in live mode, and afterwards complementing these images with ultrastructural information. Unfortunately, the term CLEM is usually associated with recent studies using state of the art high-resolution 3D-LM. However, many studies only require a rather simple correlation with low-resolution wide-field/fluorescence LM, which is illustrated for some examples (Fig. 2), highlighting the importance of coordinates facilitating CLEM, e.g. (i) finding rare cells growing in low density (Fig. 2a); (ii) finding cells with inclusions (Fig. 2b); (iii) selecting vital cells of a population (Fig. 2c); (iv) registering heterocysts of filamentous cyanobacteria, either for cross or longitudinal FIB/SEM milling (Fig. 2d); or (v) selecting immunolabeled chromosomes in metaphase (Fig. 2e).
A critical requirement for CLEM is firmly maintaining the sample in position, thus maintaining the orientation from live imaging LM to SEM. As only a minor fraction of biological specimens are inherently adherent or sticky (e.g. adherent cell cultures), it is necessary to establish procedures to immobilize a wide range of biological objects, such as single cells, cell aggregates, tissues, or tissue sections. Sample adhesion has to be stable to withstand all changes of solutions, buffers, fixatives, and solvents both by plunging or centrifugation. Finally, a prerequisite for correlative microscopy consists of embedding specimens in very thin layers of epoxy resin for precise re-localization of regions of interest (ROIs). Depending on cell size, density, etc., it is desirable to control the thickness of the sample with regards to the requirements of the scientific question. The first attempts for flat embedding were achieved by draining the resin by gravity, centrifugation, or careful blotting (Kizilyaprak et al., Reference Kizilyaprak, Bittermann, Daraspe and Humbel2014). In another study, animals infiltrated in 100% resin were removed from the resin droplet using toothpicks or pins, moved onto the substrate and drained with filter paper (Schieber et al., Reference Schieber, Machado, Markert, Stigloher, Schwab and Steyer2017). To significantly reduce the final resin layer, cells were infiltrated with 100% resin, then quickly rinsed with 100% ethanol to remove of excess resin (Belu et al., Reference Belu, Schnitker, Bertazzo, Neumann, Mayer, Offenhäusser and Santoro2016). In a recent book chapter, the technical possibilities for various embedding protocols (classical en bloc embedding and thin-layer plastification) were compared for live cell imaging of adherent cells with volume SEM using Ibidi µ-dish 500 or MatTek finder grid dishes. Minimal resin covering of cells was achieved by upright positioning of the cell substrate for draining and with polymerization starting at lower temperatures (Lucas et al., 2017).
Our aim was to enable CLEM, by avoiding any hazardous or complicated manipulations, to evolve from an expensive method into a cost-effective technology suitable for a wide spectrum of biological samples. To this end, we focused on the following aims: (i) to design and produce slides and coverslips with a variety of customized coordinates for correlative LM and SEM and FIB/SEM of both critical point dried (CPD) and flat embedded samples; (ii) to establish a labeling technique for “post-embedding”; (iii) to evaluate strategies to immobilize cells and tissue sections; (iv) to develop a filter system for “flat embedding” of large, fragile or delicate specimens; and (v) to use thick epoxy sections for high-resolution LM, TEM, and FIB/SEM tomography. Each topic was validated for at least one scientific challenge.
Material and Methods
Manufacturing of Customized Coordinates
Elevated Labels for Slides and Cover Slips
Coordinates, which are elevated and added on top of the surface of the slides/cover slips, were made by Gaßner Glastechnik GmbH (Planegg, Germany) with transfer pictures (decalcomania), glued to the slide and sintered at high temperature (Point finder; Wanner et al., Reference Wanner, Gaβner, Formanek and Greiner1990, Reference Wanner, Gaβner, Formanek and Greiner1993). AG Lasergravuren (Weilheim, Germany) produced extremely dense and small labels with sintered titanium on cover slips.
Engraved Slides and Cover Slips
Laser engravings on slides or coverslips were produced by Laser Marking, (Fischen, Germany), Grüner Laser Products GmbH & Co. KG (Munich, Germany) and AG Lasergravuren (Weilheim, Germany), according to our desired coordinate systems template.
Water-Resistant Stamp
A water-resistant stamp with a coordinate system according to our design was manufactured by modico GmbH & Co KG (Fürstenfeldbruck, Germany).
Biological Material
Chromosomes were isolated, fixed with formaldehyde, and processed as described by Wanner & Schroeder-Reiter (Reference Wanner and Schroeder-Reiter2008) and Wanner et al. (Reference Wanner, Schroeder-Reiter, Ma, Houben and Schubert2015). M. bavaricum (kindly provided by Prof. Dr. Dirk Schüler; University of Bayreuth) were fixed onto slides either by drop-cryo preparation (Wanner et al., Reference Wanner, Vogl and Overmann2008) or by high-pressure freezing as described by Jogler et al. (Reference Jogler, Wanner, Kolinko, Niebler, Amann, Petersen, Kube, Reinhardt and Schüler2011). Anabaena catenula (strain SAG 1403-1; EPSAG, Göttingen, Germany) was fixed with 2.5% glutaraldehyde in cacodylate buffer (2 mM NaCl; 2 mM MgCl2; 75 mM cacodylate; pH 7,0) or high-pressure frozen and freeze substituted before immobilization onto slides. Porphyridium purpureum was purchased from EPSAG, fixed with 2.5% glutaraldehyde in cacodylate buffer (2 mM NaCl; 2 mM MgCl2; 75 mM cacodylate; pH 7,0) and embedded into epoxy resin. Tradescantia zebrina was provided by the Botanical Garden (Munich, Germany) and fixed with 2.5% glutaraldehyde, 2 mM NaCl; 2 mM MgCl2; 75 mM cacodylate; pH 7,0. HeLa Kyoto cells were kindly provided by Prof. Dr. Heinrich Leonhardt (LMU, Munich, Germany). Cells were cultured and grown on laser marked slides and fixed as described by Luckner & Wanner (Reference Luckner and Wanner2018). Human platelets were cultured and immunolabeled by Dr. Florian Gärtner (Klinikum, LMU) (Gaertner et al., Reference Gaertner, Ahmad, Rosenberger, Fan, Nicolai, Busch, Yavuz, Luckner, Ishikawa-Ankerhold, Hennel, Benechet, Lorenz, Chandraratne, Schubert, Helmer, Striednig, Stark, Janko, Böttcher, Verschoor, Leon, Gachet, Gudermann, Schnitzler, Pincus, Iannacone, Haas, Wanner, Lauber, Sixt and Massberg2017). Breast cancer cells (SKBR3) were kindly provided by Prof. Dr. Angelika Vollmar (LMU) and fixed the same way as HeLa cells. Mouse brain tissue was kindly provided by Prof. Dr. Jochen Herms (DZNE, Munich, Germany) fixed with 2.5% glutaraldehyde in cacodylate buffer and post-fixed as described below.
Coating of Slides with Adhesives
Poly-lysine (Merck, Darmstadt, Germany)
Laser marked slides/cover slips were coated with poly-lysine according to the manufacturer’s instructions. Poly-lysine coated slides were used for drop/cryo-fixation of chromosomes (Martin et al., Reference Martin, Busch, Herrmann and Wanner1994; Wanner & Schroeder-Reiter, Reference Wanner and Schroeder-Reiter2008; Wanner et al., Reference Wanner, Schroeder-Reiter, Ma, Houben and Schubert2015), fixation of M. bavaricum (Jogler et al., Reference Jogler, Wanner, Kolinko, Niebler, Amann, Petersen, Kube, Reinhardt and Schüler2011) and Chlorochromatium aggregatum (Wanner et al., Reference Wanner, Vogl and Overmann2008).
Biobond (Science Service GmbH, Munich, Germany)
Slides were coated according to the manufacturer’s instructions and stored for weeks. For a first test, a drop of sample (cyanobacteria, diatoms, green algae, filamentous algae, biofilm) was placed on a coated slide, covered with a cover slip, and gently pressed, resulting in a thin layer of cells and providing contact with the surface of the slide for sufficient adhesion (Fig. 3a). The coverslip was removed after a few minutes and the slide was gently agitated in the buffer, medium etc. and again covered by a cover slip.
Cell-TakTM (CorningTM, New York, NY, USA)
Cell-TakTM a mussel protein (Waite & Tanzer, Reference Waite and Tanzer1981) was used for adhesion of vibratome sections of mouse brain and histological sections with a thickness up to 100 µm. Cell-TakTM was applied to slides according to the manufacturer’s instructions. The cells/tissues were placed on coated slides in the buffer, covered with a covered slip gently pressed for a proper contact of the tissue to the slide.
Bondic® (VIKU UG, Munich, Germany)
Bondic® is a viscous adhesive polymerized by UV radiation. It is waterproof and heat resistant. A drop was spread over a slide with a coverslip. Pieces of biological material were placed in the buffer on the coated area, covered with a coverslip and exposed to UV light from the backside of the slide for a few seconds.
Immobilization and LM Procedure
Cells/tissues were immobilized with suitable adhesives previously described. Samples were sealed with a coverslip and Fixogum (Marabu GmbH & Co. KG, Tamm, Germany) in the buffer, to prevent drying during LM imaging. ROIs were marked on a template copy with the same coordinates as on the slides. For standard CLEM documentation of ROIs, two to three different magnifications (objective: 5×, 10×, 40×) were sufficient for retrieval in SEM. Depending on specimen properties, bright field (BF), phase contrast (Ph), differential interference contrast (DIC), and epi-fluorescence or laser scanning microscopy (LSM) was used (Carl Zeiss, Oberkochen, Germany). Different areas were routinely documented, since (i) there can be a loss of few cells and/or damage during handling; (ii) some cells may show insufficient fixation quality or contrast in SEM among neighboring cells that are adequate in structure and contrast. LM can be repeated after thin embedding in epoxy resin, to monitor structural changes, control of contrast enhancement, and to check preservation of selected ROIs or to select new ones.
Post-Fixation by Submerging
Samples were processed, by submerging slides in buffers, fixatives, solvents, and resins, in standard staining cuvettes (Fig. 3b). The customized rOTO-protocol, was based on (Willingham & Rutherford, Reference Willingham and Rutherford1984), with 1% OsO4 (Science Services, Munich, Germany) and 1% K4Fe(CN)6 (Merck) in cacodylate buffer for 30 min, washed three times in aqua bidest., incubated with 1% thiocarbohydrazide (Merck) in aqua bidest. for 30 min, washed with aqua bidest. three times, followed by post-fixation with 1% OsO4 in aqua bidest. for 30 min. The samples were rinsed three times with aqua bidest. and dehydrated in a graded series of acetone (10, 20, 40, 60, 80, 100%), with a 1% uranyl acetate (Science Services) step in 20% acetone for 30 min.
CPD
Dehydrated samples were CPD (Polaron, Montreal, Canada). After CPD, slides were either stored in a desiccator (to prevent hygroscopic water uptake and consequent ultrastructural changes) or in little transport boxes sealed with Parafilm (Merck).
Flat Embedding in Thin and Ultra-Thin Epoxy Layers
Immobilized cells and tissues were processed and embedded on a glass slide.
Thin Embedding
Immobilized cells/tissues were infiltrated with 1:1 Hard-Plus Resin-812 (Science Services) in acetone for 15 min, 2:1 for 30 min, 75–100% Hard-Plus Resin-812 for 30 min at RT. The excessive resin was removed by draining (Fig. 3b).
Ultra-Thin Embedding
Immobilized cells/tissues were infiltrated with 1:1 Hard-Plus Resin-812 in acetone for 15 min, 2:1 (resin in acetone) for 30 min and 75–100% Hard-Plus Resin-812 for 30 min. The slides were immediately placed in a falcon tube, saturated with acetone, allowing the excessive resin to drain into filter paper at the bottom of the falcon tube for 30 min. To prevent dilution of the resin, direct contact of the slides with the filter paper, soaked in acetone, was avoided by putting a spacer (e.g. polypropylene cap) in between. As the acetone cannot volatilize, the resin concentration is not increased during infiltration and maintains its high fluidity to drain quantitatively. In some cases, when necessary, an additional centrifugation step was added (2 min; 1000 rpm). All samples were polymerized at 60 °C for 72 h (Fig. 3c).
Protected SEM Preparation by Filter System
If tissues could not be immobilized, a filter system was used (Fig. 3f). Samples were placed, with a drop of buffer on hydrophilic, acetone-resistant 13 mm ipPore Track Etched Membranes (it4ip, Belgium), with a thickness of 12 µm, a pore size of 0.4 µm and a pore-density of 1×108/cm2 to enable a sufficient flow rate by an appropriate stability. A second membrane is then placed to cover the tissue; subsequently the “membrane sandwich” is placed on top of the planar bottom part of the filter holder. The filter system is closed with the upper part of the holder, containing an O-ring (Fig. 3f). Care has to be taken that the O-ring is not contacting the “membrane sandwich” by screwing the plug. Reagents for post-fixation, dehydration, and resin infiltration were injected by 5 ml syringes (B. Braun Melsungen AG, Germany). Before polymerization, samples were transferred to slides. Draining and blotting of the excessive resin, with filter/lens paper or with a dust cleaner and monitored with a stereo lens (Fig. 3f), was effective in removing excessive resin until the samples appeared completely dry.
Thick Epoxy Sections
Classic TEM resin blocks were trimmed and sections with a thickness of 5–10 µm (depending on the cell size and density) were cut with a glass knife, then placed onto a drop of aqua bidest. on a laser-marked slide. If resins are brittle, the specimen can be heated with a blow dryer to ~50 °C to increase elasticity. While heating the slide to ~60–80 °C, the sections soften and stretch to their original size, which can be controlled while viewing with a stereo lens. Once the water droplet has evaporated, the sections stick to the slide.
Mounting and Conductive Coating
Coverslips were used in their entirety, whereas glass slides were scored with a diamond scriber (Ted Pella Inc., Redding, CA, USA) and a ruler and fractured into appropriately sized pieces, to reduce the surface area of the glass and consequently potential for charging during SEM investigations (Fig. 3g). If in-lens SE or in-lens energy selective backscattered electron (EsB) detectors were needed for highest resolution, the samples were cut to smaller pieces to allow short working distances (WD) in the range of 1–2 mm. Pieces were mounted with conductive silver colloid (Plano, Wetzlar, Germany) onto standard aluminum stubs (Plano) (Fig. 3g). Specimens were sputter coated with layers of a few nanometer (3–5 nm) of platinum (Balzers AG, Liechtenstein) for high-resolution SEM at low kV (Fig. 3g). For immuno-SEM and energy dispersive X-ray (EDX) analyses, specimens were carbon coated (3–5 nm) by evaporation (Cressington Scientific Instruments UK, Waterford, UK). If specimens were prepared for FIB/SEM-tomography, a carbon coating of 10–20 nm was used for both conductivity and protection, forming a glass-like, very stable conductive layer which is still transparent for higher energetic BSEs. For high-resolution SEM at low kV (0.8–1.8 kV), slides were carbon coated before cells/tissues were grown or immobilized, to enable conductivity without any coating (Table 1). The thickness of the glass slides (approx. 1 mm) or coverslips (precision coverslips: 0.17 mm) has no influence on SEM imaging if well grounded with silver colloid.
SE, secondary electrons; EsB, energy selective backscattered electron; QBSD, quadrant backscatter electron detector; EDX, energy dispersive X-ray; CPD, critical point dried; FIB, focused ion beam.
High-Resolution SEM
Samples were imaged with a Zeiss Auriga 40 FIB/SEM workstation operating under SmartSEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). For re-localization and a rough correlation of LM images with SEM micrographs, the WD has to be large (10 mm) and the kV high (5 kV) to ensure a sufficiently low magnification with acceptable low geometrical distortion. Although surface details are best monitored at 1 kV with the In-lens SE detector, correlation with LM micrographs sometimes need as much depth information as possible, gathered by the EsB detector (at 1–5 kV) or with the 4-quadrant backscatter electron detector (QBSD) at higher accelerating voltages (5–30 kV). Thin layers of resin then become transparent and laser marks are clearly visible. Using BSE signals, a larger aperture (60 µm) is necessary for a sufficient signal to noise ratio, which does not influence resolution at low and moderate kV. The high current mode (a feature of some Zeiss SEMs) is of benefit if the depth of focus is of importance: high current mode increases the active probe current by a stronger activation of the condenser lens. The resulting smaller angle of convergence increases the depth of field, which is important when imaging entire cells (with a height of 10–20 µm), which is not common with SEM in standard configuration.
High-Resolution FIB/SEM
Cells and tissues were milled and imaged with an Auriga 40 FIB/SEM workstation operating under SmartSEM or Atlas-3D (Fibics Incorporated, Ottawa, ON, Canada). Ion beam currents of 50 pA–10 nA (depending on the stability of the resin) were used. FIB/SEM milling started right in front of the target structure. Depending on the desired resolution image, voxel sizes between 2 nm and 10 nm in x/y were chosen. A milling rate that yield 2 nm slices allows the adjustment of the z resolution in 2 nm steps at any time during the FIB/SEM run. Due to metallic rOTO impregnation of the tissue, conduction with colloidal silver, carbon coating by evaporation, and an optional Pt-deposition upon the ROI, charging was completely avoided. As rOTO impregnation provides a strong material contrast, short exposure times down to 17 sec/image (3072×2048 pixel) could be achieved. The surface of the glass slide, as part of the image, serves as a reference for the xz. plane. For additional alignment in yz, reference lines were milled with a 50-pA beam next to the target region.
3D-Reconstruction
The resulting data sets were aligned using AmiraTM (Thermo Fisher Scientific, Waltham, MA, USA), first automatically with the module “align slices” and corrected with the “shear” function. The quality of the alignment has to be verified by the references, the slide (xz) and the added reference lines (yz) and, if necessary, corrected manually. Image stacks were segmented and reconstructed in AmiraTM (Thermo Fisher Scientific) and/or processed with a volume-rendering algorithm (volren) for direct visualization. 3D reconstructions/correlations were performed with AmiraTM.
Results
Linking Samples to Coordinates
We first tested several methods to label coordinates on glass slides and coverslips. All these labels were clearly visible in LM optical modes (bright field, DIC, phase contrast) as well as in SEM due to their topographic contrast (Fig. 4). The labels of the classic point finder (a sintered decalcomania with silk-screen printing), with a height of ~10–15 µm, yielded the strongest BSE signal, due to the lead content of the paint (Fig. 4a). Depending on the downstream applications, the composition of this paint can be modified if desired.
We found that laser engravings generated much finer lines. Upon closer inspection of fractured slides, the label appeared as a slight engraving outlined by an elevated border, deriving from either sintered TiO2, a carbon compound, or both, which were used for focusing the laser and for the energy transfer (Figs. 4b–4e). In this regard, the term engraving is a bit misleading. For CLEM and FIB/SEM of a thin embedded specimen, elevated labels were required to poke out of the thin layer of resin (Figs. 4a, 4e). In general, when an inverted microscope was used for LSM, laser marked coverslips were preferable (Figs. 4d, 4e).
Although the fabrication of fine coordinates is limited in terms of line width and accuracy, the quality of the labeling is, in practice, of secondary importance. In fact, the unique edges of imperfect markings or engravings can be used to facilitate fast correlation. The “mesh size” of the labeling can be adjusted depending on the size of the samples, but in all cases, the coverage of the coordinates is below 15%. For samples already flat embedded on a slide or cover slip, or in situations where the labels were no longer visible due to the formation of a surface layer or hidden by epoxy resin, we used a water-resistant stamp to print a customized coordinate system on top of the specimen (Figs. 4 and 3f). After documentation and carbon coating for conductivity, the coordinates of the stamp were still visible in the SE-image by topographic and material contrast, which enabled a re-localization of the previously selected ROIs (Fig. 4f).
Sample Immobilization
Proper immobilization is crucial to link the biological samples to the coordinate system of the substrate. This connection has to be rather strong to withstand the manipulations and buffers used during processing for SEM and FIB/SEM microscopy. Depending on the size, shape, and adhesive properties of the sample, a variety of “glues” can be applied for immobilization. For example, laser marked slides coated with poly-L-lysine, are routinely used for drop-cryo fixations of prokaryotic and eukaryotic microorganisms. Typically, these cells are abundant, so even the loss of a high percentage of cells is mostly inconsequential.
As a general rule, the larger the cells, the lower the yield of stably fixed cells remaining after sample preparation. As a result, stronger glues are required for such larger samples. In this regard, Biobond showed a rather strong adhesion to cells in addition to being easy to handle and moderate in price. Biobond was successfully used to immobilize a wide range of mobile algae. In contrast, filamentous cyanobacteria forming slime sheets were initially improperly immobilized with Biobond. However, after choosing strains which do not form sheets, these cells were successfully immobilized.
Next, Cell-TakTM, derived from a mussel protein, is a very strong adhesive. Cell-TakTM showed stronger adhesion properties than Biobond, which were beneficial for fixing larger tissue sections to slides. However, immobilizing thin or fragile tissue sections over their entire surface, without any folds, can be difficult. As soon as the tissue sections are in contact with the mussel protein, they will immediately adhere. If any folds occur due unevenly laying the tissue on the slide, the tissue cannot be flattened without disruption. As Cell-TakTM has to be applied for each specimen individually, the adhesive layers vary in thickness and are not as thin and uniform compared with Biobond coating. This may be a disadvantage when investigating samples such as cellular protrusions, flagella, or cilia. These structures would be difficult to distinguish at higher magnification due to the uneven topography of the glue.
Bondic®, developed as an adhesive, can be used to immobilize samples in aqueous conditions. This adhesive is polymerized by radiating the contact area for a few seconds with UV light from the backside of the slide. It is an inexpensive and strong adhesive. However, the layers are rather thick and therefore only suitable for large and sturdy samples, such as those that can be handled with forceps.
For flat embedding of delicate and fragile tissues sections, we developed a customized filter system for tissue sections, which mechanically stabilizes the sample. To this end, the tissue sections are placed between two filter membranes and screwed in a filter holder, thereby ensuring that no significant forces perturb the geometry of the sections during the entire fixation and embedding process (Fig. 3f). In addition, only small amounts of post-fixation solutions are needed, due to the small volume of the holder. Importantly, large vibratome sections remain flat, even during dehydration, as they are mechanically stabilized, which is essential for FIB/SEM. Finally, excess resin is then removed, and samples are polymerized on laser marked slides (if coordinates are needed). A variety of samples are quite suitable for processing using the customized filter system:
∙ large histological sections;
∙ samples, which cannot be immobilized by adhesives;
∙ samples, which are very fragile; and
∙ samples, which have to remain flat during preparation/dehydration.
Choosing the Right Embedding
Depending on the scientific question, it is often desirable to control the thickness of the resin layer for flat embedding. In this regard, we demonstrate adjusting the embedding thickness with HeLa cells (Fig. 5). With the portfolio of procedures ranging from simply draining the resin, to centrifugation and infiltration in an acetone-saturated chamber, the thickness of the resin layer can be adapted from ultra-thin to thin embedding. These procedures were suitable for a broad spectrum of adherent or immobilized cells and tissues (Figs. 5b–5e). In general, a thin resin layer was advantageous for the stability of the FIB-SEM run (Fig. 5h). In contrast, ultra-thin embedding was preferable for CLEM of inner cellular features (e.g. centrosomes, kinetochore), which had to be re-localized with high precision in FIB/SEM (Figs. 5c, 5f). An efficient way of producing very thin layers of resin consists of embedding the specimens in 75–100% (resin/acetone). Since samples are infiltrated in a saturated acetone atmosphere (Fig. 3c), the resin concentration is not increased and maintains its high fluidity. After an appropriate time of infiltration, the excess resin/acetone mixture is drained either by gravity or centrifugation, resulting in an extremely thin resin layer after polymerization (Figs. 3c, 5a, 5c, 5f, 5i). Minor milling artifacts at the surface, which are the result of an extensively textured cellular surface, can be ignored, if the target area is located within the cell and if these artifacts do not influence the ROI itself (Fig. 5f). For correlative LM and SEM or FIB/SEM, ultra-thin to medium-thin embedding is beneficial, since SEM and LM images (DIC, bright field) were almost identical qualitatively and their correlation was very precise (Fig. 5a), comparable with CPD cells (compare: Fig. 5b with 5c), but with a substantial gain in resolution using the EsB signal, and visibly less curtaining during milling (Figs. 5f, 5g). If thin embedding is desired, the correlation is impeded because cells are partially hidden within resin droplets (Fig. 5h). Nevertheless, the identification of selected cells was still carried out by superimposing LM and SEM micrographs. For experiments relying on cellular surface information or looking at externalized vesicles such as exosomes, thin embedding is preferable (Fig. 5h). In all cases, regardless of the resin thickness, references (glass slide and reference lines) were clearly visible in each tomographic image (Fig. 5h).
X-ray analysis allows the estimation of resin thickness (before FIB/SEM) by increasing the kV until the silicon signal, derived from the glass slide becomes prominent (Figs. 5i, 5j). The X-ray signals of carbon, nitrogen, phosphorus, osmium, and uranium of ultra-thin and thin embedded HeLa cells at 25 kV correlated with the outlines of nearly all cells (SE image) present in the mapping area. By passing through the covering resin layer of thin embedded cells (in contrast to ultra-thin embedded cells), the primary electron beam loses a lot of energy and spreads in diameter, resulting in a blurry appearance of the cells (Fig. 5j).
Care must be taken during SEM and preparation for FIB-milling: thin resin layers on glass slides are sometimes very sensitive to the electron beam. Higher magnifications, used for focusing and correction of astigmatism, rapidly lead to the formation of “bubbles” in the resin, which look striking, but did not influence further milling. Low magnifications and using an appropriate kV are recommended for SEM and FIB adjustment. Once the FIB/SEM milling process has started, the energy input is concentrated onto the block face, and the risk of “bubbles” is negligible. For thin specimens (1–5 µm) a very low ion beam current (50 pA, as used for FIB imaging) is recommended.
Examples for Analytical SEM and FIB/SEM and Possibilities for CLEM
Chromosomes: Immuno-Labeling—from LM to FIB/SEM
The topography of isolated cell organelles or single cells can be investigated with high resolution, after application of a thin metal coating (3–5 nm platinum), using a short WD (1–2 mm) and with the in-lens SE detector at 0.8–1.2 kV. However, the platinum coating is incompatible with metal-based staining such as platinum blue for DNA or immuno-labeling with small gold colloids (5 nm). Even the thinnest platinum coating will prohibit detection of metal-based staining or gold-labels with the EsB-detector (Figs. 6a3, 6b4). Carbon coating can be used for conductive coating if higher currents are required, but with a dramatic loss of high-resolution topographic information gathered at 1 kV. However, for a high-resolution analytical investigation of isolated biological structures such as chromosomes, carbon coating of the substrate (glass slides) before applying the sample is ideal since: (i) the problem of charging can be minimized and even eliminated, either at low kV or due to the conductive carbon layer; and (ii) the rather dark background in the BSE image enables high-resolution SE and high contrast BSE imaging at very low kV (0.8–1.5 kV).
Carbon-coated slides remain translucent after coating, therefore enabling LM to select ROIs beforehand. Since the re-localization of small biological structures such as mitotic chromosomes is not trivial, a linked reference coordinate system is critical. For example, the mitotic index for spelt is very low (2–15%), and therefore a previous selection in of ROIs in LM was essential (Fig. 6a1). FluoroNanogoldTM-labeled antibodies against alpha-tubulin were located at fibrillar bundles at the centromeric region of the mitotic chromosomes (Figs. 6a2, 6a3). Analytical investigations of metal impregnations at different depths of the sample were carried out by varying the negative grid voltage of the in-lens energy selective BSE detector for high resolution (Fig. 6).
To detect labeled structures located inside a biological structure, the kV must usually be increased. Typically, charging begins e.g. at 2–3 kV, but disappears at higher kV as the electrons penetrate the specimen and reach the conductive carbon layer. In this case, the system must be operated at two different accelerating voltages: 1 kV to image the surface topography and surface-located labels (in-lens SE detector, EsB detector) (Fig. 6a2, 6a3) and 10 kV for high-resolution imaging of labels in the interior of the sample (QBSD). Additionally, the low BSE-yield of carbon prevents interfering BSE signals from the substrate. To demonstrate this, holocentric chromosomes of Luzula elegans were stained with DAPI (for DNA) and with gold-labeled antibodies against a phosphorylated variant of Histone H2A (for centromeres). These chromosomes were first visualized with phase contrast (Fig. 6b1), and with fluorescence microscopy (Fig. 6b2) and then re-located in SEM (Fig. 6b3). Gold-labeled antibodies (anti-Histone-H2A) were then visibly localized within the centromeric groove (Fig. 6b4). To verify the spatial distribution of the gold labels, the specimen was carbon coated before FIB/SEM milling, to avoid charging during milling. As a result, gold labels were clearly visible on both sides of the chromosomes (Fig. 6b5).
CPD samples of chromosomes can also be used to study DNA distribution with FIB/SEM. However, CPD may cause shrinkage and formation of cavities. To avoid dehydration artifacts, embedding in Moviol, a water-soluble resin, is beneficial, and offers a smooth block face which yields the highest resolution (Fig. 6c). The BSE signal of the Pt-stained chromosomes showed the global distribution of the DNA (Figs. 6c4, 6c5).
For correlative LM and FIB/SEM microscopy, samples are commonly embedded in a resin block together with the substrate. The substrate is then removed after polymerization by thermal shock, thereby leaving the cells in the resin. However, if the experiment involves examining how the cell contacts the substrate, for example, to analyze attachment or migrating activity, then the substrate cannot be removed. Therefore, in such cases, ultra-thin or thin embedding directly on the slide is required. With immuno-fluorescence microscopy, it was demonstrated that human platelets migrate and pile up on the adhesive substrate together with any bound particulate material. This occurs when actomyosin-dependent traction forces overcome substrate resistance (Gaertner et al., Reference Gaertner, Ahmad, Rosenberger, Fan, Nicolai, Busch, Yavuz, Luckner, Ishikawa-Ankerhold, Hennel, Benechet, Lorenz, Chandraratne, Schubert, Helmer, Striednig, Stark, Janko, Böttcher, Verschoor, Leon, Gachet, Gudermann, Schnitzler, Pincus, Iannacone, Haas, Wanner, Lauber, Sixt and Massberg2017) (Fig. 6d1). Once removed, fibrinogen is transported toward the center (pseudonucleus) of migrating platelets, remaining on the platelet surface, mainly within invaginations of the open canalicular system. After LM (Fig. 6d1) platelets were thin embedded, re-located in SEM and covered with two platinum protection layers by ion beam deposition (Fig. 6d2). Additionally, a carbon layer was implemented between these two layers for milling high contrast tracking lines for FIB/SEM microscopy using Atlas3D hard- and software. These tracking lines enabled correction of astigmatism and focus (autostig and autofocus) during the FIB/SEM run (Fig. 6d3). Escherichia coli cells were collected and accumulated at the pseudonucleus together with the fibrin(ogen)-forming bundles of several bacteria (Fig. 6d3). A 3D-rendered FIB-SEM stack of the platelets demonstrates the accumulation of the E. coli via fibrin (Fig. 6d4).
Microorganisms: From fast Results To High Resolution
For non-adherent microorganisms, proper immobilization is a critical requirement for cost-effective FIB/SEM high-resolution experiments. In this regard, poly-L-lysine coated slides are widely known to provide adequate adhesion of prokaryotic cells. In order to demonstrate the potential and widespread applicability of fast, analytical investigations with FIB/SEM, we examined a mixed culture, containing magnetobacteria. While EDX in SEM is not commonly used in biology, it remains a very effective way to reveal for, example, metallic inclusions etc. Typically, only a minor fraction of M. bavaricum is found within an enriched mixed culture. Cells, immobilized by drop-cryo fixation, were CPD. ROIs containing magnetobacteria were then selected in LM by their characteristic size and shape (Figs. 7a1, 7a2). In order to retrieve the selected ROIs in SEM, we applied a stamp with a reference coordinate system on top of the CPD sample (Fig. 7a3). Chains of magnetosomes were then visualized by BSE at higher voltages (Fig. 7a4) and EDX mapping clearly confirmed and highlighted their iron content (Fig. 7a5). Together, these data allow statistical quantification of the number of magnetosome chains, their length, and their position.
There are, however, some limitations to using CPD specimens immobilized onto glass slides for EDX analysis, due to the strong signal from the glass (Si, O, and their numerous additional components). However, if beam damage is not a severe problem and if the elements have their energy lines in a suitable range, element analysis can be performed at low kV (1–3 kV) (Fig. 7a5).
We then obtained the three-dimensional distribution and architecture of single magnetosomes with FIB/SEM (Fig. 7b). CPD samples were post embedded with resin for higher resolution, compared with non-embedded samples (compare: Fig. 7b2 with 7c2). The cellular matrix was filled with epoxy resin, with resulted in a smoother block face. Since the topographic contrast of BSE is reduced, the signal to noise ratio of BSE is significantly improved (Fig. 7c2). We obtained the best ultra-structural preservation and resolution of structural details when magnetobacteria were high pressure frozen and freeze-substituted (as described by Jogler et al., Reference Jogler, Wanner, Kolinko, Niebler, Amann, Petersen, Kube, Reinhardt and Schüler2011), then infiltrated in resin and spread on glass slides before polymerization (Fig. 7d1). Single cells, located in droplets of resin, still offer the possibility for post-embedding correlation with LM to identify target cells and enable directed (longitudinally or cross-sectioned) milling for efficient FIB/SEM microscopy (Fig. 7d2). Finally, structural details were reconstructed with high resolution in 3D, allowing for quantification of number and distribution of multiple cellular structures: single magnetosomes and chains, storage granules of sulfur and poly-β-hydroxybutyrate, as well as the cellular envelope and the bundle flagella (Figs. 7d3, 7d4).
Multicellular organisms: Targeted FIB/SEM
As discussed earlier, the larger the sample, the more elaborate is its immobilization, since different surface properties often require specific adhesives. Many multicellular cyanobacteria, such as Anabaena, produce specialized nitrogen-fixing heterocysts, which are organized in filaments. These filaments are characterized by a regulated developmental pattern of single heterocysts, separated by vegetative cells. We found that Biobond was sufficient for proper adhesion of Anabaena filaments to slides. With LM (DIC, fluorescence), we selected and documented different stages of the transition from vegetative cells to heterocysts, according to the coordinates of the slides (Fig. 8a1–8a3). ROIs were then easily retrieved since the appearance of the filaments was essentially identical in SEM when compared with LM micrographs (Fig. 8a4).
FIB/SEM acquisition of CPD processed samples started directly in front of the selected heterocyst, which allowed setting a precise milling direction (in this case, longitudinally) (Fig. 8a4). Since the sample is not embedded within an epoxy resin, milling a ramp to access the target region is unnecessary. Excavations between membranes gave a strong topographical contrast (Fig. 8a5) at the expense of the BSE signal and consequentially resolution (Fig. 8a6). Of note, the large gap between the cell wall and the membrane of the heterocyst is at risk of charging. When comparing SE and BSE signals of vegetative cells, the block-face was visibly much smoother due to their dense, compact thylakoid system (Figs. 8a7, 8a8), even though, small holes still disturbed the image quality (Fig. 8a8).
Conveniently, the auto-fluorescence of Anabaena filaments was maintained after ultra-thin embedding and high-pressure freezing (Figs. 8b1, 8b2). This allows easy verification of the developmental status of heterocysts, and as a result, the quality of FIB/SEM was significantly improved (Figs. 8b4, 8b5). This improvement takes place even though the space between the cell wall and protoplast of the heterocysts remained empty (Fig. 8b4). We obtained the best results when thicker layers of resin were applied (Fig. 8c). A small ramp had to be milled in front of the target, however, it is clearly smaller compared with classic embedding protocols (Figs. 8c2, 8c3). As a result, single thylakoid membranes were clearly resolvable (Fig. 8c4), which then enabled striking 3D reconstructions, offering insights into the complex architecture down to 2 nm voxel sizes (Fig. 8c5).
Tissues: From LM to FIB/SEM
Immobilizing tissue sections to a slide stabilizes the sample during SEM preparation. This, in turn, keeps the sample flat and allows thin embedding directly on the substrate, without losing orientation. In our hands, tissues were adequately immobilized with Biobond or Cell-TakTM. To look further into this, we imaged a piece of Tradescantia epidermis, which inherited several stomata (Fig. 9a1). To determine the position of organelles such as nuclei or chloroplasts, we imaged the sample at higher magnification with different optical modes (DIC, fluorescence, Figs. 9a3, 9a4). Removal of excess resin is of primary importance. To this end, if tissues are tightly fixed (e.g. Cell-TakTM), a moderate centrifugation (500 rpm for 5 min) will remove most or all of the resin obscuring the surface. After polymerization, the SEM image was similar to the LM image (Fig. 9a2), which enabled an easy and fast correlation of these two images (Fig. 9a5). This demonstrates that the target area had sufficient topographic contrast to recognize the stomata over the entire epidermis (Fig. 9a2). This, in turn, allowed for precise and directed milling (longitudinal or cross-sectioned) with FIB (Figs. 9a6, 9a7) and subsequently 3D reconstruction (Fig. 9a8).
In neuroscience, FIB/SEM became an important tool for ultrastructural studies addressing connectomics of the entire brain. For example, vibratome sections of brain tissues are commonly used to study neuronal processes (Fig. 9b1). Slices can be rather large (>1 cm2) or very fragile, which makes adequate immobilization onto slides particularly challenging. In this regard, the filter system described earlier facilitates a gentle post-fixation, dehydration and infiltration of resin until the sections are in a stable condition (Fig. 3f). Superimposition of LM and SEM micrographs using the characteristic outline of the brain section was generally sufficient for a rough correlation (Figs. 9b2, 9b3). Natural markers like blood vessels and nuclei, detectable in both LM and SEM images, served as fiducial markers to re-locate ROIs within the SEM (Figs. 9b4, 9b5). The distinctive shapes and sizes of structures at the surface, or sectioned natural landmarks (e.g. blood vessels appear as channels or large holes, nuclei as dark dots), facilitated a subsequent superimposition of light and electron micrographs and to define the target area in x/y direction. The depth of the target dendrite within the tissue was determined by aligning block face images with the corresponding micrographs of the LSM stacks and triangulation of the respective landmarks (Fig. 9b6). A rough 3D reconstruction of potential target dendrites within the ROI (Fig. 9b8) revealed the selected dendrite by its unique spine arrangement (Figs. 9b7, 9b9).
Epoxy Sections for economic FIB/SEM
Several aspects favor the use of thick epoxy sections for FIB/SEM: (i) parts of biological specimens cannot be immobilized for various reasons and have to be embedded conventionally within resin blocks; (ii) an archive of samples already exists, which may be used for FIB/SEM years after initial preparation; (iii) samples were investigated with TEM and corresponding 3D stacks are desired of the same ROI. We found that resin sections mounted on laser marked slides was an elegant way for FIB/SEM milling of target structures embedded in resin blocks. The high potential and efficiency of thick sections was demonstrated for the red algae Porphyridium purpureum (Fig. 10a) and for SKBR3 breast cancer cells (Fig. 10b). ROIs were selected with bright field, DIC or phase contrast (Figs. 10b1, 10b3, 10b5). As sections are typically in the millimeter scale (feed size), the correlation was easily carried out by merging LM and SEM micrographs. At moderate kV (3–5 kV) Porphyridium purpureum were detected with the BSE signal (Fig. 10a4). Cells that overlapped in LM and SEM were located at the surface and already inter-sectioned. Cells visible in LM but not in SEM were intact within the section (compare Fig. 10a3 with 10a4). We then selected intact cells and directly targeted them with FIB/SEM, omitting the need for a large trench to find whole cells by chance (Figs. 10a5; 11h). Stages of mitosis, infected cells, cells undergoing apoptosis etc. were found with different optical modes (BF, Ph, DIC) (Figs. 10b1–10b5). We selected a mitotic cancer cell, then milled and imaged it with FIB/SEM and finally reconstructed it in 3D (Fig. 10b). Ten-micron sections appeared a bit more sensitive to the ion beam. It is thus recommended to reduce the ion beam energy, which is not a problem as milling speed is generally much faster than imaging time. In addition, the use of a harder mixture of epoxy resin (EMS Hard-Plus Resin 812) is recommended.
Discussion
High-Resolution Analytical SEM for CLEM
Correlative LM combined with SEM and FIB-SEM is of fundamental importance to biomedical research. However, in order for this technique to evolve to a routinely used method, reproducible, efficient and cost-effective procedures need to be further developed. In this regard, compared with TEM, SEM has not reached the widespread use in bio-sciences that one might expect given its high resolution and potential. The potential of analytical high-resolution SEM techniques remains largely unexplored for many scientists starting out with FIB/SEM tomography. Published protocols may suggest that FIB/SEM milling is a stand-alone technique for correlative microscopy. However, we would argue that FIB/SEM milling is only one of the many facets of high-resolution SEM. Fully exhausting the benefits of all SEM capabilities is a prerequisite for efficient routine correlative microscopy. The right handling of WD, high voltage, the use and variation of different parameters (chamber SE, in-lens SE, in-lens EsB, QBSD, apertures, high-current mode) mean that SEM can be a versatile and powerful tool for a wide range of applications (Scala et al., Reference Scala, Pasquinelli, Martegani and Laschi1985; Bozzola & Russell, Reference Bozzola and Russell1991).
Correlation of light and EM as a method became substantially more elaborate with the introduction of serial block-face sectioning. Now, a 3D-LM data set with precise reference coordinates serves a basis (Lucas et al., Reference Lucas, Günthert, Gasser, Lucas and Wepf2012; Karreman et al., Reference Karreman, Hyenne, Schwab and Goetz2016; Cheng et al., Reference Cheng, Shami, Morsch, Huynh, Trimby and Braet2017; Lees et al., Reference Lees, Peddie, Collinson, Ashby and Verkade2017; Russell et al., Reference Russell, Lerner, Burden, Nkwe, Pelchen-Matthews, Domart, Durgan, Weston, Jones, Peddie, Carzaniga, Florey, Marsh, Gutierrez and Collinson2017). For standard EM preparations, specimens have to be cut into small pieces, which is accompanied by a significant loss of the 3D-context. Even after oriented embedding, correlation can still be exquisitely difficult. The basic problem is that, in contrast to 2D, we are typically unable to correlate volumes without computing. Even our eyes do not see in 3D but rather interpret spaces by 2D images on the retina. The simple postulate is: correlate in 2D and make the specimen suitable for it.
Which geometry of biological samples is desirable or restrictive for CLEM? The main limitation is in the FIB/SEM operation. The maximum milling depth is about 100 µm, but due to curtaining in at these depths, a more practical depth is around 50–60 µm. Specimens within that range are suitable for LM. Thus, if cells or tissues are naturally within this range (bacteria: few µm; HeLa cells: 20 µm; diatoms/algal cells 5–100 µm), then they simply have to be immobilized onto a slide. Depending on the sample, however, this can be quite challenging. If larger tissues are used (e.g. mouse brain with a volume of approx. 1 cm3), the classic way of cutting small cubes with a feed size of 1 mm may result in an astronomic number of pieces (e.g. ~1000, each with the risk of severe damage), which should be fixed and embedded numerically. Cutting the same volume with a vibratome into 50–70 µm sections reduces the number of individual slices to 150–200. Compared with 1 mm3 cubes, these slices have, due to their minor thickness, several advantages: (i) they are suitable for all LM modes (bright field, DIC, fluorescence, CLSM); (ii) fixation and contrast enhancement is faster and more consistent due to more permissive diffusion conditions; (iii) the entire slice can be investigated with SEM at high resolution; (iv) each ROI defined in LM can be re-localized by simple triangulation, as LM and SEM images match perfectly (Fig. 9b).
The Right Coordinate System
It is undisputed that a robust coordinate system is a solution for any correlative microscopy technique. This implies that instead of the structural details, the images of the coordinates in LM and SEM are initially correlated. This correlation is trivial, but only if the labels are clearly visible in both microscopy modalities (Fig. 5a). Today, complete solutions are commercially available and generally based on the same idea: selecting an ROI in LM, storing the coordinates, switching to SEM and recalling the coordinates (Liv et al., Reference Liv, Zonnevylle, Narvaez, Effting, Voorneveld, Lucas, Hardwick, Wepf, Kruit and Hoogenboom2013; Brama et al., Reference Brama, Peddie, Wilkes, Gu, Collinson and Jones2016; Schorb & Sieckmann, Reference Schorb and Sieckmann2017). This is a valuable improvement if a set of instruments from the same manufacturer is used and trimming, mounting etc. does not alter the specimen. In practice, however, LM and SEM systems from different manufacturers, which do not intrinsically share a coordinate system, are often used for CLEM. To this end, inherent labels on the specimen are most beneficial for investigating the same specimen on different systems, for example when changing from LM instruments (LSM for fluorescence and DIC) to SEM or using micro-CT or X-ray microscopy. With different labeling methods covering a broad range of requirements for different applications, correlation with any microscope should be possible. Since the coordinate system is a 2D pattern, superimposition and only linear scaling or rotation is required—if at all. Finally, any image distortion is instantly recognized.
Immobilization and Flat Embedding
A prerequisite for any flat embedding of cells, cell aggregates, and tissues is their immobilization in a fixed position relative to the coordinate system. Immobilization of different biological objects, capable of withstanding the entire EM procedure, including exchanging fixatives, solutions, washing steps, and dehydration in ethanol or acetone, will always be challenging. From a variety of available adhesives, we found two that particularly stand out. First, Biobond had very good adhesive properties for prokaryotic cells, larger eukaryotic cells and cell aggregates (Fig. 8). Since the adhesive coating is in the nm range, the evenness of the slide is maintained, which is important for high-resolution SEM, e.g. of cells with flagella or filopodia. Second, Cell-TakTM is the strongest adhesive and therefore the best choice for larger tissue sections. As Cell-TakTM is preferentially spread with a coverslip, the thickness cannot be precisely controlled, which limits its application in SEM for high-resolution topography of cell adherent structures (Fig. 9a). Although the chance for successful immobilization of any objects is on 50%, flat embedding is still possible by alternative strategies. If live-cell imaging is dispensable or is not possible (e.g. high-pressure freezing of non-adherent cells), samples can be processed until infiltration with resin, spread onto slides with coordinates and embedded ultra-thin or thin (Figs. 3d, 7d, 8b, 8c). CLEM is still possible as fluorescence can surprisingly still be detected for a variety of objects, although much weaker, after HPF, FS, and embedding (Fig. 8b). This signal provides practical information e.g. about vitality (vegetative cells of Anabaena catenula) or stages of cell differentiation (heterocysts of A. catenula) for a directed and efficient milling (Fig. 8). It is generally attractive to embed samples into resins which maintain fluorescence such as Lowicryl (Kukulski et al., Reference Kukulski, Schorb, Welsch, Picco, Kaksonen and Briggs2012). However, it must be determined experimentally whether this rather soft resin is stable enough for high-resolution FIB/SEM. If samples are (i) available only in few individuals; (ii) fragile; (iii) or have to be kept flat during fixation and dehydration (histological sections), the presented filter system is of great benefit (Fig. 3f).
CLEM is limited, but still possible for histological sections, as their characteristic shape and surface features are retained and can be examined after thin/ultra-thin embedding with LM and SEM (Fig. 9). The necessity and advantage of thin embedded samples became a topic of discussion within the last years, with the increasing demand of FIB/SEM in biosciences. A few protocols are published for flat embedding in resin by draining, blotting, centrifugation with the primary aim to reduce the resin layer to a minimum (Kizilyaprak et al., Reference Kizilyaprak, Bittermann, Daraspe and Humbel2014; Lucas et al., 2017; Schieber et al., Reference Schieber, Machado, Markert, Stigloher, Schwab and Steyer2017). In practice, however, the aim is not an ultimately thin epoxy resin layer, but rather to control the thickness, depending on the samples and the scientific question. The main obstacle to this control is that the viscosity of resin rises significantly on the seconds to minutes timescale during spreading into thin layers, resulting in non-reproducible thickness. The removal of excessive resin by ethanol (Belu et al., Reference Belu, Schnitker, Bertazzo, Neumann, Mayer, Offenhäusser and Santoro2016) is a good attempt but bears the risk of uncontrolled reduction, which is critical, when prokaryotic cells should be embedded in a resin layer of only a few microns thick. By keeping the slides in an acetone-saturated chamber (Fig. 3c), the fluidity of the resin is retained, until an even spreading is achieved. By changing the parameters (surface properties of slides or cover slips, resin concentration, draining by gravity, centrifugation, exposure time), a suitable thickness of resin can be achieved for individual samples after a few test runs. We illustrate the multiple possibilities for immobilization, fixation and thin embedding of various specimens for CLEM in a flow chart (Fig. 12).
Thick Sections for Bridging TEM-Tomography with FIB/SEM
When comparing TEM- and FIB/SEM tomography, the excellent TEM resolution in xy cannot be reached with SEM due to physical and geometrical considerations (Giannuzzi, Reference Giannuzzi2004). Section thickness for TEM-tomography, however, is limited to ~200–1000 nm, ideal for prokaryotic cells and cellular structures as the endoplasmic reticulum, but not for the complex 3D architecture of entire eukaryotic cells (Ercius et al., Reference Ercius, Alaidi, Rames and Ren2015). TEM-tomography followed by FIB/SEM tomography is an attractive solution to combine the highest resolution with very large volume information. This works reliable if a 200–400 nm section from a resin block is used for TEM-tomography and the following 10 µm section is used for FIB/SEM of the same ROI, which can be repeated several times in cycles. Within a very large 3D volume (e.g. an entire cell), high-resolution 3D details can be implemented. The potential of thick serial sections for FIB/SEM is impressively demonstrated by Hayworth et al. (Reference Hayworth, Xu, Lu, Knott, Fetter, Tapia, Lichtman and Hess2015).
Defining Space
Three orthogonal planes define a cube, typically achieved by FIB/SEM milling, resulting in an image stack. After milling, the defined planes are not maintained due to specimen drift, image distortions etc. (Schaffer et al., Reference Schaffer, Wagner, Schaffer, Schmied and Mulders2007; Boergens & Denk, Reference Boergens and Denk2013; Šedivý & Jäger, Reference Šedivý and Jäger2017; Storm et al., Reference Storm, Beckmann and Rau2017). Therefore, an alignment of each image stack is necessary, which requires absolute fiducial markers for reference. The block face image represents the xy plane. If image stacks of resin blocks are automatically aligned (without references), the alignment is optimistically assumed to be correct, as it cannot be verified easily, except if substructures of known geometry (e.g. spheres) are present in abundance. The generation of references is not trivial for resin blocks. However, thin embedded samples provide the second reference plane in xz automatically, given by the surface of the slide/coverslip (Figs. 5f–5h; 11). The mandatory third reference plane can be easily produced, by milling parallel “lines”, which represent de facto vertical planes through the resin, ending orthogonally in the glass slide (Figs. 5h; 11j). In fact, most FIB/SEM image stacks require drift correction, and thus reliable references are necessary.
Independent of the method used for alignment of a FIB/SEM stack (Kreshuk et al., Reference Kreshuk, Straehle, Sommer, Koethe, Cantoni, Knott and Hamprecht2011; Cardona et al., Reference Cardona, Saalfeld, Schindelin, Arganda-Carreras, Preibisch, Longair, Tomancak, Hartenstein and Douglas2012; Saalfeld et al., Reference Saalfeld, Fetter, Cardona and Tomancak2012; Schindelin et al., Reference Schindelin, Arganda-Carreras, Frise, Kaynig, Longair, Pietzsch, Preibisch, Rueden, Saalfeld, Schmid, Tinevez, White, Hartenstein, Eliceiri, Tomancak and Cardona2012), the accuracy can be validated by the reference coordinates and adjusted manually, which is likely necessary for long series (Han et al., Reference Han, Zhang, Gao and Murphy2018). Without references, any changes in volume geometry cannot be recognized or corrected (Storm et al., Reference Storm, Beckmann and Rau2017). There are promising examples of intelligent software which aim to learn each step of alignment by mimicking the human approach (Sommer & Gerlich, Reference Sommer and Gerlich2013; Kraus & Frey, Reference Kraus and Frey2016; Kan, Reference Kan2017). These programs, however, require substantial computing power and are still far away from being routinely applicable to large data sets.
Cost-Effectiveness Considerations
If state of the art techniques is established as routine methods, cost-effectiveness becomes an important consideration. FIB/SEM will be always time-consuming, due to the enormous number of images for high-resolution tomography. Numerous scientific questions lead to results to some extent by chance, for instance when pellets of microorganisms are milled (Fig. 11g). In this regard, obtaining a longitudinal section through a heterocyst of cyanobacteria between vegetative cells, at the right stage and in the right orientation, is like winning the lottery. With flat embedded cyanobacteria filaments this can be achieved routinely with all controls, using different strains and culturing conditions (Fig. 8).
For questions involving eukaryotic cells or tissues, several efficiency aspects have to be considered. For example, for a HeLa cell in metaphase (spherical, with a diameter of 20 µm), a cube with a 20 µm feed size has to be milled. Then, by setting the section thicknesses to 10 nm, 2000 sections are needed. Under best conditions, with rOTO contrast enhancement (Seligman et al., Reference Seligman, Wasserkrug and Hanker1966; Willingham & Rutherford, Reference Willingham and Rutherford1984) and using the in-lens SE-detector, the exposure time for an acceptable block face image can be around 15 s. This results in roughly an 8 h exposure time for the whole cell, thereby possible within a day. Classical glutaraldehyde/osmiumtetroxide (Palade, Reference Palade1952) fixation, however, gives a much weaker contrast. If using the EsB detector, an exposure time of one minute or more is expected. Thus, the FIB/SEM experiment becomes a 2-day venture with at least one overnight session, bearing risks of the instability of the system, heating of the Ga-emitter and loss of information during the restart of the milling procedure. For a HeLa cell, milling time is much less than the exposure time. However, for cross sections of C. elegans, the milling time increases dramatically, especially when low ion currents (100–200 pA) have to be used. FIB/SEM investigations increase to several days or even weeks with costs of several thousand dollars per run.
The costs for FIB consumables (Ga-emitter, apertures, service costs) can be reduced to 50% simply by flat embedding due to omitting a ramp and reducing the volume to be milled exclusively to the ROI (compare: Fig. 11g with 11i). Beside economic aspects, an important benefit of flat embedded samples, compared with established procedures, is (i) saving time; (ii) the possibility of setting the milling frame as precisely as desired for cross or longitudinal section; and (iii) starting close to structural details documented before in LM, e.g. cellular inclusion, centrosomes etc.
Summary
We developed an easy and broadly applicable procedure, consisting of several technical improvements of relevant scientific investigations, including the whole range of simple correlative, high-resolution LM/SEM microscopy, with immuno-labeling, flat embedding for FIB/SEM and post-embedding investigations of specimens (Fig. 12). Since every sample has its own handling requirements and limitations, several adjustments based on sample properties and scientific question are offered (Fig. 12). Defining and maintaining coordinates of a target structure is the most important aspect for re-localization in SEM. With sample preparation, coordinate labeling, and with the right conductive coating, the analytical potential of the SEM with all detectable signals (SE, BSE, EDX) is of great advantage for correlative investigations.
Acknowledgments
The excellent technical assistance of Silvia Dobler, Jennifer Grünert, and Cornelia Niemann is gratefully acknowledged. The authors are much obliged to Joel Ryan for carefully revising the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.