3View: A New Perspective for Cell Biologists

By Christel Genoud and Simon Galloway, Gatan UK

3View is a new product and technique from Gatan which is set to revolutionize microscopy in many fields. This is due to the relative ease with which high spatial resolution ultrastructure can be obtained from extensive volumes. The prototype for 3View (SBFSEM, serial block face imaging in the SEM) developed by Denk and Horstmann1 at MPI Heidelberg has pushed the spatial extent, and acquisition speed of 3D ultrastructure at relatively high spatial resolution. Furthermore, unlike other techniques the resolution in the Z direction can be close to the X,Y resolution, and it does not degrade with depth. As the technique is largely compatible with accepted EM specimen preparation for biological sciences, 3View is poised to transform microscopy in a variety of disciplines.

3View has generated considerable excitement in Denk and Horstman’s research field of neuroscience. This challenging research area places stringent demands on 3D extent, integrity and resolution. With this proven capability, 3View is ideally placed to impact related research interests whether in STEM cell research, embryology, pathology, etc.

For example, one leading research group in cell biology proposed an exciting challenge for Gatan to solve. They want to study the entire nucleus of culture grown cells in 3D, in one experiment and at magnification to resolve the double nuclear membrane. The challenge was therefore not just the automated acquisition of ultrastructure using the 3View, but an acceptable, low risk protocol for specimen preparation.

Mammalian cells are challenging samples for electron microscopy for many reasons. The most important perhaps is that the cells are kept in cultures in order for them to grow. This places constraints on the SEM microscopy preparation technique. To grow, cells must be attached to a support medium such as fine glass circles. Cells on the glass grow to form a monolayer of cells that touch each other. The glass slides are submerged in a medium that feeds them. The way they react to different solutions put in their growing medium can be studied. For example cell response to putative drugs, compounds found in the environment, anoxia, growth factors, etc.

Various labelling methods can be then used on them to highlight the morphological modifications of the cell.The use of confocal microscopy has revealed the structure of the nucleus of cells in three dimensions. As this technique has matured the limits of the spatial resolution have been reached as determined by the physics of standard or 2-photon confocal microscopy (also pioneered by Denk2).

Electron microscopy allows this fundamental barrier to be broken and thus allows the study of ultrastructure of the nucleus with higher resolution. The traditional approach of cutting and examining TEM sections can be used. However 3View offers a much faster solution, especially as collecting and examining serial sections in the TEM is a very skilled and laborious task. With 3View, the cutting and imaging takes place in-situ in the FESEM, and the backscattered electron image is taken from each fresh cut of the block face. In this way, with good specimen preparation, the 3D resolution does not vary thoughout the experiment.

The cells have been stained in order to enhance the image contrast from the electron irradiation. The cells are also resin embedded to provide a support medium that can be cut with a diamond knife. However, embedding cells attached to a glass slide is non trivial. In addition, the fact that the cells are fixed and embedded on the glass slide directly means that once the glass support has been removed, the cells are exposed at the interface. This surface must not be cut or damaged during the preparation of the sample or the preparation for the acquisition. (Figure 1).

Figure 1. The embedding of cell cultures in order to observe them in electron microscopy. (A) Cells are grown on glass slides as a supporting medium. (B) Cells attached on glass slides are fixed. (C) Cultures are resin embedded on the glass slide. (D) A crucial step consists in separating the resin and the glass slide. The cells are thin and at the surface. (E) 3View image acquisition from the block face starts from the surface of the cells.

A small piece of this culture / resin ensemble is attached to the 3View specimen holder, as shown in Fig.2.

Figure 2. A culture grown cell sample with top surface intact is now attached to a 3View specimen holder.

Figure 3a shows the whole cell culture including cell membranes and a faint “shadow” of the cell nuclei which is more visible at slightly higher magnification in Figure 3b. The cells of interest can then be targeted. To avoid unwanted electron dose into the region of interest, fine focusing of the electron beam is normally performed at a slightly different location. Once an acceptable field of view, pixel density and scanning speed is established by the microscopist, the software user interface simply requests a final specification of the number of cuts and cut thickness to perform and the experiment proceeds automatically. During acquisition each new image can be viewed live, and the most recent series of images can be viewed without disturbing the acquisition. As data sets can be very large, a rebinned 3D thumbnail also evolves, so that the progress of the whole of the experiment can be viewed during acquisition.

Figure 3c shows the full montage of images recorded at 50nm cut thickness. In this particular experiment, the plane of the glass resin interface was not completely planar with the knife cut plane (which is the focal plane of the microscope). This doesn’t interfere with the quality of the results, but simply means that the first few cuts are visible at the top left hand corner of the field of view. This gives additional confidence that the integrity of the top surface is maintained using this protocol. With this method we have been able to acquire images of every fresh block face between the knife touching the surface until the cells have disappeared and only resin remains, as seen in the final image in the thumbnail montage.

Figure 3. Example of images of cells in cultures acquired with 3View. (a) An image of the pyramid has been taken at low magnification. Cells are visible as dark areas. Light area is the empty resin. The surface of the block is trapezoid to optimize the cutting. (b) A region of interest to be cut in series has been chosen at higher magnification. The image shows the intact surface of the sample as detached from glass (dusts and uneven surface is visible). The outside cell membrane appears at the surface of the resin. (c) Montage of the 120 images obtained. Each plan is 50 nm thick (for details, see movie M1)

Click here for movie M1 in Quicktime (MOV) format (12.3 meg)
Click here for movie M1 in WMV format (1 meg)
Click here for movie M1 in AVI format (12.3 meg)

These images are aligned as recorded and require no post processing for this purpose. They can be visualized as movies with each frame slicing through the specimen in the plane of the knife (M1), or reconstructed in an alternative orthogonal configuration. (M2). These movies have been rebinned to a lower pixel resolution for convenience.

Click here for movie M2 in Quicktime (MOV) format (20.5 meg)
Click here for movie M2i in WMV format (6,7 meg)
Click here for movie M2 in AVI format (20.5 meg)

The original images are at high pixel resolution and thus it is possible to zoom into regions of interest. (fig 4). Gatan’s software allows data to be exported for further processing, 3D rendering and segmentation task by a variety of 3rd party packages, but this subject is not treated in this short article.

Figure 4. Example of the resolution achieved with 3View in this particular experiment. (A) Original field of view. (B) detail of (A). The resolution and pixel density is sufficient to distinguish the double membrane of the nucleus and the structural details of the nucleolus.

Gatan would like to acknowledge Dr S and J Fakan (Centre of Electron Microscopy, University of Lausanne) for their kind help and cooperation in providing specimens.

References

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