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Blockface imaging

SEM blockface imaging - the principle

In SEM blockface imaging, specimens are processed using protocols very similar or identical to methods used for TEM sample preparation, but are imaged by scanning electron microscopy. Following fixation, samples are stained heavily with various heavy metals prior to embedding in resin. Samples are then mounted on aluminium SEM stubs either as ultrathin-sections placed on glass or plastic coverslips (rather than TEM grids) or the entire resin block can be fixed on a stub. Both specimen types need to be sputter-coated with a thin layer of carbon to avoid charging and are then imaged in the SEM in backscatter mode. Inverting the B/W contrast of the blockface images then produces results very similar to those obtained by TEM.

 

Green algae - Cosmarium subtumidum - Myriam Goudet, Howard Griffiths, Physiological Ecology Group, Dept. of Plant Sciences, University of Cambridge.

Cosmarium subtumidum
Map of a culture of green algae (Cosmarium subtumidum) revealing the morphology of the pyrenoids, the main actors of the algal carbon concentrating mechanism. Pyrenoids improve the efficiency of photosynthesis by delivering carbon dioxide to RuBisCo, an enzyme concentrated at the heart of this micro-compartment. Surrounded by a typical starch sheath, the pyrenoid is pervaded by an intricate network of microtubules.

 

 

Advantages of blockface

The advantages of SEM blockface imaging over TEM are apparent: it is possible to view areas much larger than would even fit onto a standard TEM grid and the specimen is free of grid bars, which always obscure part of the sample in TEM; it allows to gain an overview of a sample not possible by TEM and makes it easier to find a particular area of interest. However, the useful magnification of blockface imaging is presently limited to about 8,000 – 10,000x. The FEI MAPS software on our Verios 460 SEM can be set up for acquisition of image tiles in an automated fashion producing large specimen maps, which can be zoomed at any region of interest.

 

Drosophila melanogaster egg chamber - Marzia Munafo, Greg Hannon Group, Cancer Research UK, Cambridge.

Block overview of a Drosophila ovary
Block overview of a Drosophila ovary. A map was set up in the area delineated by the green rectangle.

 

Drosophila ovary
High-resolution map of a Drosophila melanogaster egg chamber: the area of the nurse cells to the left and the regular row of follicle cells to the right enclosing the developing oocyte. The areas in the coloured insets are shown in more detail below.

 

Drosophila Ovary Red
Nurse cell nucleus showing the numerous pores in the nuclear membrane.

 

Drosophila ovary green
Follicle cells with large nuclei and mitochondria of various shapes.

 

Drosophila ovary blue
Boundary between the developing oocyte (left) and follicle cells (right).

 

 

Stereology approaches

The capability of acquiring large overview maps and imaging sub-areas at higher resolution in a defined location can also be advantageous for quantification of specific structures by stereology.

 

Hannah Somerfield/Dr Adrian Butcher, Professor Giovanna Mallucci’s Group, UK Dementia Research Institute, University of Cambridge – Quantification of synapses in a mouse model of neurodegeneration

Mouse Hippocampus
Overview map of part of a mouse hippocampus. A mapping area was defined 100 µm from the pyrimidal cell bodies of the hippocampal CA1 region.
 

Mouse Hippocampus HR
High resolution map of the synaptic region of the hippocampus defined by the green square in the overview map.

 

Mouse Hippocampus Detail
On zooming in on the high-resolution map (green inset), synapses characterized by docked pre-synaptic vesicles and post-synaptic density can be identified; six synapses are highlighted by the green dots.

 

 

Compatibility with TEM 

The similar specimen processing makes SEM blockface imaging and TEM truly complementary techniques. It is possible to view a large specimen block by SEM to find a rare or small region of interest, which can be delineated using the SEM linescan function. After selectively trimming this area in the ultramicrotome, ultrathin sections can be viewed by TEM at much higher magnification and resolution than achievable by SEM. Alternatively, smaller specimen areas can be used for acquiring serial sections for array tomography and 3D reconstruction, or the SEM-mounted resin blocks can be used for imaging by FIB-SEM. CAIC is not yet set up for array tomography or FIB-SEM, but is planning to implement these techniques in the near future.