|
|
SBFSEM: New frontier for
|
By Christel Genoud and Joel Mancuso, Gatan Inc.
Initially targeting the neuroscience, 3View™, the serial block face scanning electron microscope (SBFSEM) presented by GATAN explores new biological applications. Histology, being an essential tool of biology, is the study of thin-sectioned tissue, using a microtome as a way of describing anatomy at a microscopic level. Understanding of the spatial organization of specific tissues requires the knowledge of 3D organization. Traditionally, this information has been acquired by either serial sectioning or confocal microscopy. Unfortunately each technique has its limitations. Serial sectioning is labor intensive and prone to human error, while confocal microscopy has limited resolutions and observation is restricted to labeled structures. SBFSEM overcomes these limitations by, improving resolution of unlabeled and labeled tissue, and automating the acquisition of large volume serial images.
In the case of SBFSEM specimen preparation, two key considerations are the level of contrast shown by the specimen, and the resilience of the embedding resin to the electron beam radiation. For histological samples, after primary fixation, the tissue has been post-fixed in osmium tetroxide with ferrocyanide to help with known contrast issues. In addition, en-bloc staining using uranyl acetate and other staining agents further enhance the contrast of some organelles. Epoxy based resins (e.g. epon, durcupan) are superior when compared to acrylic resins. An optimal recipe has been developed to withstand beam current conditions necessary for SBFSEM (Table 1.)
Table 1. SBFSEM Expoxy Resin Recipe.
| SBFSEM EPON 812: |
| 37 ml glycid ether 100 |
| 25 ml DDSA (Dodecenylsuccine acid) |
| 20 ml MNA (Methyl nadic anhydrrde) |
| 1.3 ml BDMA (Benzldimethyl amine) |
Specimen preparation including block trimming is advised because it can influence the stability of the stacks, especially when automated cuts of extensive acquisition times are required. An embedded tissue is glued to the top of a specimen holder specific to 3View™. Fortunately for histological samples, tissues are large and easily trimmed to obtain a rectangular or trapezoidal surface of approx 300x400 up to 900x900 microns. It is critical for the two sides parallel to the knife are trimmed at 45 degrees. The other two sides which are almost perpendicular (creating the classic trapezoid block face shape) are trimmed at 90 degrees. It’s important to keep region of interest centered to accommodate any drifting of the region of interest due to slight misalignment.
|
Movie 1: Mouse lung 3D volume, 3D model created in DigitalMicrograph 3D Visualization software plug-in. This data set contained over 800 50nm consecutive sections collected using 3View. Model dimensions are 100 x 150 x 20µm. Orginal image was 2k x 2k, recorded at 1000x at 5keV collected with a FEI Quanta 600 with an attached 3View |
Click here to download movie in AVI format - 170 Meg
Click here to download movie in WMV format - 6 Meg
The first histological example we would like to highlight using SBFSEM is lung tissue. Movie 1 demonstrates how serial block face imaging in conjunction with Digital Micrograph can produce a 3D volume model. In this sample this model was constructed with over 800 sections collected unattended overnight. With no misalignment for all the sections, creating an accurate and useful model is quick and straightforward when compared to other 3D modeling techniques. Lung tissue is an excellent tissue to render in 3D due to the large amount of empty space in the sample. This kind of reconstruction could be for example useful to study at ultra structural level the tissue alterations occurring in mouse model of lung cancer in which the lung tumor cells are fluorescent.
| Movie 2: Mouse skin tissue imaged at 300x, Orginal 2k x 2k image
allows for both cellular and structural examination. Over all field of
view is 500µm x 500µm. 1000 50nm sections were imaged creating a total
volume 50um thick. The data set was processed in Digital Micrograph
using 3View plugins to create a 3D model. Data collected with a FEI
Quanta 600 with an attached 3View. Click here to download movie in MPG format - 3 Meg |
Being a highly organized tissue skin is an excellent example to demonstrate imaging and acquiring large volumes of tissue as demonstrated in Move 2SBFSEM has the ability to image large field of view at high resolution allowing cellular level (high magnification) and structural level (low magnification) investigations and at the from the same image without interfering grid bars present in a low magnification TEM image. Studies of mouse skin using SBFSEM, highlights views of the layers of the epidermis, the epidermal derivatives as well as the layers of the dermis. Skin 3D organization is crucial for its protective role as well as its nervous functionality. Disorganization of this structure leads to pathologies such as skin tumors. Skin biopsies are systematically sent to a laboratory of dermatopathology that fix, cut and study the extension as well as the type of lesion removed. It is especially critical to assure that the totality of the lesion is removed in the case of a tumor. 3View could be a good complementary tool to study the extension of the lesions at a higher resolution to better understand the processes involved in tumor infiltration.
| Movie 3: Mouse kidney slice. Image dimensions 250 µm x 120 µm, 862
slices of 50nm with a total volume 43.10um. Orginal image was 2k x 2k,
recorded at 600x at 5keV, collected with a FEI Quanta 600 with an
attached 3View. The data set was processed in Digital Micrograph using
3View plugins to create a slice player movie. Click here to download movie in AVI format - 92 Meg Click here to download movie in WMVformat - 6 Meg |
These examples are of healthy organization of tissues. However, it is also interesting to examine pathological states where there is a disorganization of the classical structure of a tissue. 3View will not replace the classical histological techniques but is a new tool allowing the user to reach high resolution in an extended volume.
All the examples illustrate the intimate link between the 3D ultrastructure and the functions of tissue. In some cases, revealing 3D ultrastructure from SBFSEM will provide new evidence that will either corroborate or question established biological models of healthy and pathological tissue. It may also provide key data to resolve known biomedical issues.
