Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues
© The Author(s). 2016
Received: 18 May 2016
Accepted: 29 November 2016
Published: 12 December 2016
Imaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips.
To ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution and 100 nm (the section thickness) resolution in z.
By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we can address volumes from single cells to complete organs. In our first example, a cell population isolated from zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of cells at decreased resolution we can define the percentage at which different cell types are present in our preparation. With the second example, the root tip of cress, we illustrate how combining information from intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data need to be stored, handled and eventually analysed.
Our custom-designed substrate holder allows reproducible generation of section libraries, which can then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict, that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.
KeywordsArray tomography Serial sectioning Section libraries Hierarchical imaging Large volume 3D reconstruction
In view of the recent success of super resolved fluorescence light microscopy or nanoscopy, as it is also called by one of the Nobel awardees , the question arises how relevant electron microscopy (EM) will be for the future of the life sciences. When it was introduced not quite 100 years ago it was not exactly a method suited to image entire cells or even complete model organisms at nanoscale resolution. However, new developments in volume EM [2, 3] are challenging that statement.
There are several ways to create volume EM data: The blockface methods, serial blockface scanning electron microscopy (SBFSEM: ) and focussed ion beam scanning electron microscopy (FIBSEM, reviewed in ), are well established in the field. Here the surface or blockface of a sample embedded in a resin block, is alternately imaged and removed in a cyclical manner in a SEM. Both methods are destructive, consuming the sample while it is being imaged. For SBFSEM this can lead to the necessity of imaging very large areas, in extreme cases the whole blockface, at rather high resolution, because it is not possible to rescan interesting areas later. In this way huge data sets (cf. ) are produced which may contain only few regions with really interesting events or substructures.
Another possibility to explore the third dimension with EM is the array tomography (AT) approach where arrays of ultrathin serial sections are deposited on large, solid substrates and imaged either in a light microscope (LM) or in a SEM. The method was originally introduced for multiplexing immuno-staining by repeated stripping and re-labelling of the section arrays in order to map synaptic connections in brain [6, 7]. In the neurosciences field, that pioneered all volume EM techniques (reviewed in ), variations of the original method are quite common, also extending it to SEM imaging (reviewed in [2, 9]). However, applications in cell and developmental or even general biology are rather scarce up to now [10–12]. One advantage of this method is its potential for hierarchical, targeted imaging, which we will illustrate with examples in the present paper. AT also allows correlative or conjugate  approaches, when substrates amenable to LM are used. To this end we developed a tool that helps to reliably create arrays of sections on a number of different substrates, suitable for SEM as well as for different modalities in LM.
Custom-built substrate holder as prerequisite for reliable retrieval of multiple ribbons
Additional file 4: Movie S1 Substrate holder in action. (MP4 16804 kb)
Multi-scale imaging of large volumes on arrays of ultrathin serial resin sections
Imaging with electrons relies on heavy metals to deliver good signals, so we used samples as typically prepared for transmission electron microscopy. During the preparation, osmium and uranium were incorporated into the block and the sections were additionally post-stained with uranium and lead. Initial manual imaging of arrays in the SEM proved very tedious, recording 70–100 sections for the 3D reconstruction of a small cell easily required several hours of rather concentrated work. More recently, we were able to test as early adopters the newly released ZEISS Atlas 5 Array Tomography platform (Carl Zeiss Microscopy, GmbH, Oberkochen, Germany). The platform consists of a scan generator that can create images of up to 32k x 32k, which in turn may be stitched together to even larger “regions” using the software package supplied with it. The software takes over the command of the microscope and can also be used to image every section of the array (section sets) and subsequently predefined regions of interest (ROIs) within every section (site sets). This solution allows hierarchical imaging at different resolution levels in an automated manner, as explained below.
Additional file 6: Movie S2 Hierarchical imaging on an array of 200 serial sections. (MOV 18307 kb)
Nanomorphomics in a cell population – organelle inventories and more
Additional file 7: Movie S3 3D representation of cytotoxic immune cell from zebrafish spleen. (MOV 5255 kb)
Additional file 8: Movie S4 Image stack of complete neutrophilic granulocyte with segmentation. (MOV 1744 kb)
Additional file 9: Movie S5 3D representation of neutrophilic granulocyte from zebrafish spleen. (MOV 5178 kb)
Additional file 10: Movie S6 3D representation of basophilic granulocyte from zebrafish spleen (MOV 4839 kb)
Different cell types in a FAC-sorted population from zebrafish spleen
Apart from studying cell morphology at nanoscale resolution, arrays can also be used for quantification and statistics, an attribute not commonly associated with EM. The FAC-sorted population from the lymphoid gate of spleen investigated here was sorted with a rather wide gate to increase yield. The pellet shown in Fig. 3a consisted of about 50,000 cells, the yield from the spleens of five adult fish. Analysis of such a small pellet at that nanoscale resolution would be very difficult by other means. The different cell types can be counted, theoretically in the whole pellet if one would cut it up completely. In practice, sufficiently high numbers of cells can be counted by choosing individual sections far enough apart in z to exclude counting a profile from the same cell twice. For identification of a cell type, intermediate resolution (here a series imaged with 60 nm pixel size) is sufficient: Once a whole cell of a given type has been reconstructed in 3D at high resolution, characteristic features will help to identify the cell type in a single section at lower resolution. Examples are the prominent granules of the granulocytes or the characteristic cogwheel shape of the cytotoxic cell. In this manner 239 cells were counted on three different sections (cf. Table 1).
Tackling tissue – polarity in a root tip
Additional file 11: Movie S7 Zooming in to slice 36 of a cell in the cress root calyptra. (MOV 17394 kb)
Additional file 12: Movie S8 Zooming in to slice 159 of a cell in the cress root calyptra. (MOV 11655 kb)
Additional file 13: Movie S9 Zooming in to slice 227 of a cell in the cress root calyptra. (MOV 11852 kb)
Based on the AT approach introduced in 2007  we propose an easy access workflow for multi-scale hierarchical imaging applicable not only to model organisms with their dedicated genetic tools, but to many types of samples, even unique ones. Some preliminary, technical details of this workflow were already presented as abstracts to a specialized microscopy audience [16, 17].
Our custom-built substrate holder is a relatively low cost attachment – which can be retrofit - to a common ultramicrotome, an instrument available in virtually every EM lab or facility. One of the first substrate holder devices was introduced in 1964 by Behnke & Rostgaard , consisting of a stand with a cantilever and a clamp mounted on the free end of that cantilever. The clamp can hold a pair of forceps, which in turn holds the TEM grid. A rack and pinion drive between cantilever and clamp allows moving the substrate longitudinally. Very similar devices have been presented over the years [19, 20]. With only one or two degrees of freedom the adaptability to fit an ultramicrotome setup with a rotated knife is insufficient. This is the main disadvantage of these devices. To overcome this limitation Meyer & Domanico  introduced a device that is attached directly to the knife or the knife holder. This device has always the same orientation as the knife. It supports one TEM grid and the lift out movement can be motorized. Because of its focus on TEM grids it is not intended to use other substrates. Other supporting devices for TEM grids not needing any mechanical parts are also described: One idea is a modified knife boat to hold the grid under the water . It has also been described how to attach the grid on the floor of the knife boat next to the knife edge . All these modifications are limited to TEM grids, too.
The latest device published is designed for cryo-ultramicrotomy . It consists of two micromanipulators, each offering a three-way movement. One micromanipulator holds the forceps gripping the TEM grid, the other the conducting fibre for manipulating the sections. This device is designed only for one specific microtome and does not offer the adaptability mentioned before.
With our device we have an element of freedom in planning our experiment since we have a slide-sized carrier onto which we can mount a wide variety of different substrates – in principle from TEM grids to glass coverslips or silicon wafers. We also have high flexibility for sample orientation within the block since the substrate holder can be aligned with a rotated knife, which was not possible for any of the previous devices.
The ATUMtome [14, 25], an automated sectioning device using a carbon-coated Kapton tape to pick up sections, is a rather complex device with constraints reducing the field of application. It does not have e.g., the option to use a glass substrate, which will facilitate super resolution LM  and also correlative imaging . With the ATUMtome it is difficult to collect thick (1–5 μm) sections e.g., for histology, because ultrathin sections adhere much better and the rolling of the tape may lead to loss of thick sections. With our device it is no problem to produce and pick up ribbons of 1 μm thick sections.
The possibility to section physically allows very high z-discrimination in a standard wide-field fluorescence microscope down to 50–70 nm, depending on how thin the sections can be made.
Since post-staining of arrays can be done with exactly the same reagents as used in traditional TEM imaging, high metallization of samples, which is necessary for the blockface methods SBFSEM and FIBSEM, is not required. We were also able to successfully apply this workflow to human pathology samples prepared according to standard protocols with osmium as the only heavy metal in the block (not shown).
Manual imaging of arrays in the SEM at low voltage is possible, but very time consuming. Here automated imaging, specifically in combination with hierarchical imaging is a decided advantage. Contrary to the blockface imaging methods (SBFSEM and FIBSEM nanotomography), where just one imaging cycle is possible, AT allows revisiting ROIs and imaging them at different resolutions. Precious or unique samples are preserved, albeit in a “sliced-up” version. This is not the case with SBFSEM and FIB nanotomography where the samples are consumed by the process.
In addition, targeting specific structures within a tissue or rare events in a population of cells, such as e.g., identifying immunological synapses in a coculture of cytotoxic cells and cancer cells  can be realized rather easily on arrays. Medium resolution images are sufficient to screen whole pellets for the desired events. Then ROIs are only placed on the sections displaying these and imaged at the resolution required to analyze the corresponding structures in detail. For the blockface methods, much more complicated procedures such as laser branding and CLEM (correlative light and electron microscopy) are necessary to target the volume of interest in the whole block .
Dimensions of volumes, data, and acquisition time
image size (μm)
# of sections
imaging time total
spleen - whole pellet
387 × 163
60 × 60 × 100
spleen - several cells
30 × 22
5 × 5 × 100
root - cross section
246 × 246
60 × 60 × 100
root - one cell
30 × 28
5 × 5 × 100
If higher resolution in z is required, FIBSEM nanotomography on selected sections is an option we are currently exploring .
Another point illustrated by our range of examples is the relationship between physical volume size, resolution, data size and imaging time (Table 2). For example imaging of a whole root cross section (ca 250 × 250 μm) over 24 μm (240 sections) with 60 nm pixel size took 28 h and produced 4.1 GB of data. At this resolution only the larger organelles within the cells were visible. Increasing resolution to 5 nm pixel size to allow detection of all membrane-bound organelles and ribosomes increased imaging time for just one cell (ca 30 × 30 μm image size, again 240 sections) to 52 h with 8.1 GB of data. Microscope settings were comparable for both datasets (cf. Methods section). To image all of the 30 cells in the central column would have taken 65 days, if that would have been productive is another question. The combination of information obtained from the intermediate resolution data with information obtained from a representative volume imaged at high resolution was already sufficient to extract enough facts for building a tentative model of those cells’ polar organization.
To ensure reliable retrieval of ribbons of serial sections for AT from the boat of a diamond knife we introduce a substrate holder with 7 degrees of free movement specifically designed for that purpose. Using this we are able to deposit up to two hundred sections densely packed in an ordered way on an area the size of a 22 × 22 mm coverslip. When creating such arrays on substrates amenable to LM they can be imaged with very good z-discrimination even in an ordinary wide-field fluorescence microscope. Arrays on silicon wafers were imaged in a hierarchical way in a SEM using a software platform for automated imaging. Hierarchical imaging is an easy way to target rare events or substructures within a larger context. Adapting image collection in the SEM resolution-wise to the question being investigated can help to reduce the amount of data produced. Finally, combining both imaging modalities opens the way to large volume correlative approaches.
Arabidopsis roots were high pressure frozen, freeze substituted and embedded in Lowicryl HM20 as described .
Immune cells were isolated from the spleens of five adult zebrafish, chemically fixed and embedded in epoxide resin as described previously .
From cress seeds germinated for 3–4 days on wet filter paper the roots were cut off and immersed in 1% glutaraldehyde in 50 mM cacodylate buffer at 4 °C over night. After 4× 10 min washing in buffer they were postfixed in 1% OsO4 in cacodylate for 4 h at room temperature, followed by further washes, 2× 10 min in buffer and 2× 10 min in double-distilled water, they were block-stained over night at 4 °C with 1% uranyl acetate in double-distilled water. Next steps were: Further washing, 4× 10 min in double-distilled water; dehydration in a graded acetone series of 25%, 50%, 75%, and 2× 100% for 15 min each; infiltration in Spurr’s resin for 45 min each in 25%, 50%, 75% resin and over night in 100% resin at 4 °C. Before embbeding in fresh resin in BEEM capsules, 100% resin was exchanged once and kept for several hours. Resin was polymerized at 60 °C for 1 d.
Concept of the custom-built substrate holder
The holder is based on the supporting hand concept. It allows the operator to position the substrate in the boat accurately while optimising the waterline between water and substrate, which depends on the substrate material used and the contact angle. After positioning of the substrate the operator gains one hand free for other purposes. The substrate can be positioned in a wide range and even a knife rotation around the vertical axis of up to +/− 10° can be handled. To meet the +/− 10° knife rotation a lateral coarse positioning of the holder base with a travel range of +/− 25 mm has been integrated (coarse translation axes #1 and #2, see also Additional file 2: Figuer S2). The rotation of the substrate clamping unit around the vertical axis can be realised using rotation axis #3. Axis #3 allows an endless turning and is also used to rotate the holder mechanism out of the knife work space e.g., when changing the knife for trimming. The substrate position in the knife boat can be changed using axis #4 for off-centre movement (side-ways) and axis #5 for vertical positioning of the substrate (longitudinal positioning of the waterline). The substrate water surface angle can be set with axis #6 and finally the longitudinal movement of the substrate towards the knife is realised with axis #7. The lifting process after pinning all ribbons or sections at the substrate is realised using axes #5 (vertical lifting) and #6 (substrate rotation towards horizontal, see also Additional file 5: Figure S4).
Axis travel ranges
Base coarse positioning 1
+/− 25 mm
perpendicular to table front side
Base coarse positioning 2
+/− 25 mm
parallel to table front side
Substrate vertical rotation
large offset from substrate position
Substrate off-center positioning
+/− 10 mm
side-ways along knife edge direction
Substrate vertical positioning
+/− 10 mm
Substrate lowering and lift off movement
Substrate angular rotation
angle between water surface and substrate
Substrate longitudinal positioning
+/− 25 mm
in substrate plane towards/away from knife edge
Producing arrays of sections
Polymerized resin blocks were trimmed and the leading and trailing edges of the blockface coated with a mixture of 30% glue (Pattex, Henkel; Germany) in xylene to stabilize the section ribbons. Serial sections, usually 70 nm to 100 nm thick, were cut either on a UC7 ultramicrotome (Leica, Germany) or a Powertome PC (RMC Boeckeler, USA) using a Jumbo knife (Diatome, Switzerland). A custom built handling device helped to place several ribbons on small pieces of silicon wafers or ITO-coated glass coverslips (Optic Balzers, Liechtenstein). Arrays produced from methacrylate resin were stained with 1 μg/ml propidium iodide (Sigma-Aldrich, USA) in distilled water over night at 4 °C. Arrays from epoxide resin were post stained with uranyl acetate and lead citrate as described before .
Recording image data
Fluorescence images were recorded in a Cell Observer (Carl Zeiss Microscopy GmbH, Germany) wide-field fluorescence microscope equipped with a mercury arc lamp (Illuminator HXP 120 V) and a rhodamine filterset.
Imaging of arrays with electrons was done in a Crossbeam 540 (Carl Zeiss Microscopy GmbH, Germany), a field emission SEM featuring a double condensor system. Imaging conditions were: 1.5 kV, a beam current of 811 pA, ESB (energy-selective backscatter) detector, grid at 1000 V, 25.2 microseconds dwell time. Using computer-assisted tools in the newly released ZEISS Atlas 5 Array Tomography platform, serial sections were imaged at multiple resolutions: First the whole array (“region”, mosaic of about 70 tiles) was imaged automatically at 1000 nm image pixel size. Then serial sections were recorded automatically at 60 nm pixel size (“section set”). On these section images interesting cells or groups of cells were selected for further high-resolution imaging (“site sets”). These ROIs were automatically imaged over a range of 50–250 serial sections at 5 nm pixel size using a large single (up to 32 k × 32 k pixels) frame for each site.
Image stacks recorded with ZEISS Axiovision (Carl Zeiss Microscopy GmbH, Germany) or exported from ZEISS Atlas 5 Array Tomography were cropped and registered using the stackreg plugin  or TrakEM2  in Fiji .
Subsequent volume rendering and segmentation were performed with the Amira software package (VSG/FEI, USA). Movies were produced in ZEISS Atlas 5 (Google Earth-like) or Amira (segmented or rendered volumes).
Fluorescence activated cell sorting
Focused ion beam
Indium tin oxide
Red blood cell
Region of interest
Scanning electron microscope
We thank Sachin Singh, Peter Chockley, and Clemens Grabher (KIT) for zebrafish cells, Carolin Bartels and Lisa Veith for sectioning.
HEiKA funded the initial phase of substrate holder development in the framework of its Advanced Imaging Platform. This work was supported by grant FKZ13GW0044 from the German Federal Ministry for Education and Research, project MorphiQuant-3D. None of the funding bodies was involved in the research or in writing the manuscript in any way.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
IW, AH, UG, RRS conceived and designed experiments. SH prepared samples. IW, WS, MT performed experiments or recorded data. IW, WS, AH, RRS analyzed data and prepared digital images and movies. IW, RRS drafted the article. All authors read and approved the final manuscript.
KIT has received reimbursements by Boeckeler Instruments for supply of a functional model of the substrate holder. Marlene Thaler is employee of ZEISS Microscopy GmbH, manufacturer of microscope systems mentioned in this article. In addition ZEISS offers certain solutions such as Atlas software packages for a wide range of applications in large-area, 3D and volume imaging for SEM and FIB-SEM instruments.
Consent for publication
Ethics approval and consent to participate
Research involving animals
Zebrafish handling was performed in accordance with the German animal protection standards and was approved by the Regierungspräsidium Karlsruhe, Germany (General license for fish maintenance and breeding: Az.: 35–9185.64). Plants used in this study were grown in the laboratory from commercially available seeds and are not subject to protection.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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