Scanning electron microscopy (SEM) is a an effective technique, traditionally provided for imaging the surface of cells, tissues and whole multicell organisms (see An advent to Electron Microscopy for Biologists)(Fig. 1).

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Figure. 1 mirrors a backscattered electron micrograph the daisy pollen grains.


While the result images show up to be 3 dimensional (3D), they in reality contain no depth information.

However, there are several SEM techniques that can achieve 3D information about a sample. Several of which have the right to be done on any kind of microscope and also some that require specialist instrumentation, software, or microscopes in order to accomplish that result. This article guides you with the easily accessible techniques.

Stereoscopy

Stereoscopy is the procedure by which two slightly various images space presented to every eye, producing an illusion that depth in the image. The images have the right to by coloured and merged to develop a 3D anaglyph stereogram (Fig. 2) that can be perceived with anaglyph glasses. Stereograms are produced by taking two images of the very same area, tilting the specimen 5-10° in between each image. The photos are climate tinted (red-green or cyan-maroon) and aligned. A digital model of the surface ar of a specimen deserve to be developed from stereoscopic images using specialist software, which allows z axis measurements to be made (Hughes et al., 2005). Vertical or overlapping details are regularly lost or distorted utilizing this an approach and data is limited to the surface of the specimen, however this is a great method because that obtaining topographical information. Stereoscopy deserve to be used on any type of SEM through a tilting stage.


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Figure 2 reflects a chart demonstrating exactly how to produce a stereoscopic image with a SEM. Two images are take away of the specimen with in between 5 and 10° tilt between them. The images are tinted, in this situation maroon and cyan and combined to create a 3d result that deserve to be regarded using anaglyph glasses.


Photogrammetry

Photogrammetry is the procedure of recording a collection of overlapping pictures that have the right to be used to reconstruct the object gift imaged. The photos usually covering 360° that the sample from several different angles (Fig. 3). This have the right to be replicated making use of an SEM by transforming and tilting the sample to photo as lot of the surface as possible. At least 50 photos are forced to construct a decent model and this can be time consuming in one SEM. This an approach is feasible on most SEMs making use of either a hand-operated or an automated stage with a high tilt range. Photogrammetry to produce a 3D representation of the surface ar of a sample but will not provide any information of internal structure.


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number 3 mirrors a chart demonstrating just how an image series can be caught for photogrammetry. The sample is rotated and an image captured every few degrees until the complete 360° is covered. The sample is then tilted and also the process repeated. At the very least 3 image sets should be caught at various tilt angles. The best data can be acquired from a phase that has actually a full 90° tilt range.


Serial Section array Tomography

Serial section selection tomography (Micheva & Smith, 2007) is based on similar principals come serial ar TEM yet has the benefit that plenty of thousands the sequential sections deserve to be imaged. A diamond knife is used in an ultramicrotome to produce thin part of a resin embedded sample. The sections are accumulated manually onto a substrate (e.g., a on slide or coverslip) or top top a tape using an automatic system, such together the ATUMtome. When in the SEM, software application is provided to find and also image the exact same area of attention on all of the part (Fig. 4). Benefits with this method include the capability to image big volumes the tissue v EM resolution. That is also feasible to use extr stains or antibodies on the sections for selective staining and it is feasible to revisit the sample at a later date. The main disadvantages to range tomography room that there have the right to be lacking sections or artefacts such together wrinkles in the sections gift imaged and also there are limitations in the thickness of the sections, down to in between 30 and also 40nm thick v a great sample.


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figure 4 mirrors a diagrammatic representation of 3 various 3D SEM techniques, selection tomography (left), serial block confront SEM (middle) and also focused ion beam SEM (right). Each of these techniques is a serial ar method. Selection tomography images the part after they have been developed on one ultramicrotome. Serial block face SEM catches images that the sample between removing slices using a diamond knife. Focused ion beam SEM gets rid of slices making use of an ion beam.


Serial block face scanning electron microscopy (SBFSEM)

SBFSEM (Denk & Horstmann, 2004) is also a serial ar method. However, in this method the diamond knife is situated inside the SEM. The surface ar of a resin block, comprise a specimen such together cells or a tissue, is imaged making use of backscattered electrons. The block is raised and the diamond knife removes a part from the surface before one more image is captured (Fig. 4). In this way, data have the right to be captured throughout thousands of slices. Over there is no physical managing of sections; as such section certain artefacts such as wrinkling and compression are eliminated. Alignment and registration of images is often much easier than with range tomography. The minimum feasible slice size is currently about 15nm, although in between 30-100 nm is an ext common(Peddie & Collinson, 2014), which boundaries the z resolution that can be completed in the data. The full volume that have the right to be imaged is far less than with array tomography. This is because of a limit in specimen phase movement. Because that example, the Gatan 3view system has a preferably data volume of 600 x 600 x 600 µm prior to the sample needs to be repositioned. SBFSEM is a destructive an approach and the is not possible to revisit part at a later date.

Focused Ion Beam Scanning Electron Microscopy (FIBSEM)

FIBSEM (Heymann, 2006) provides a beam of ions to ablate the specimen, developing a trench through a milled level surface top top one side. The electron beam is supplied to generate picture of the level surface (Fig. 4). The ion beam is provided to mill far a tiny layer that the surface and another image is captured. This is recurring thousands that times. FIBSEM is a destructive method and sections cannot be revisited yet multiple trenches can be made in ~ the very same sample. The field of watch is frequently smaller than with range tomography and SBFSEM because of limitations in milling time. However, the z axis resolution is far higher than v the other techniques mentioned. Slices of 3-5nm space possible, leading to isotropic voxels in the last dataset (3D pixels that have the same dimension on all axes).

3D Data

Array tomography, SBFSEM and also FIBSEM all develop 3D datasets the the interior and exterior the cells and tissues. Once images have actually been accumulated they room aligned and also data is reconstructed right into a 3D volume (Fig. 5). Indigenous this data, separation, personal, instance structures can be diverted (segmented) and qualitative and also quantitative info obtained around the specimen. Data handling and analysis can be exceptionally time spend (Peddie & Collinson, 2014, Borrett & Hughes, 2016) and also requires computers with a lot of processing power however it is worth It for the comprehensive 3D details that can be obtained.


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number 5 shows data produced using serial block confront SEM, however this procedure is likewise applicable for range tomography and focused ion beam SEM. The series of pictures is first stacked, aligned and also calibrated for the exactly section/slice thickness. The 3D data stack have the right to then be examined and also subsequent handling such as filters and adjusting the brightness and contrast have the right to be applied. The last step is to extract areas of interest making use of segmentation, wherein automated or manual choice of structures such together organelles can be made throughout many slices to create a 3D volume. The 3D volume presented is of liver tissue and red blood cells have been segmented out of the data (orange frameworks on the right).

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References

Borrett, S. & Hughes, L. (2016) Reporting techniques for handling and analysis of data indigenous serial block face scanning electron microscopy. Journal that microscopy, 263:3–9.Denk, W. & Horstmann, H. (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional organization nanostructure. PLoS biology, 2:e329.Heymann, J. A. H., M. Gestmann, I. Giannuzzi, L.A. Lich, B. Subramaniam, S (2006) Site-specific 3D imaging of cells andtissues through a twin beam microscope. . Journal of structural biology, 155:63–73.Hughes, L. C., Archer, C. W. & ap Gwynn, I. (2005) The ultrastructure of computer mouse articular cartilage: collagen orientation and implications for tissue functionality. A polarised light and also scanning electron microscopic lense study and also review. European cells & materials, 9:68–84.Micheva, K. D. & Smith, S. J. (2007) selection tomography: a new tool because that imaging the molecular architecture and also ultrastructure that neural circuits. Neuron, 55:25–36.Peddie, C. J. & Collinson, L. M. (2014) trying out the 3rd dimension: volume electron microscopy come of age. Micron, 61:9–19.

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