The blocks containing the embedded tissue were glued onto a sample stub using conductive silver paint (AGAR Scientific Ltd., Stansted, Essex, UK). All the surfaces of the blocks, except that to be studied (the top surface), were covered with silver paint to prevent charging the resin. The stubs with the mounted blocks were then placed into a sputter coater (Emitech K575X, Quorum Emitech, Ashford, Kent, UK) and the top surface was coated with a 10–20 nm thick layer of gold/palladium to facilitate charge dissipation.
The three-dimensional study of the samples was carried out using a combined FIB/SEM microscope (Crossbeam® Neon40 EsB, Carl Zeiss NTS GmbH, Oberkochen, Germany). This instrument combines a high resolution field emission SEM column (Gemini® column, Carl Zeiss NTS GmbH, Oberkochen, Germany) with a focused gallium ion beam which permits material to be removed from the sample surface on a nanometer scale. Regions of the neuropil were chosen on the surface of the tissue block for 3D analysis. A protective layer of carbon was deposited on top of the area to be analyzed using an ion beam with a 30-kV acceleration potential. Using a 10-nA ion beam current, a first coarse cross-section was milled as a viewing channel for SEM observation. The exposed surface of this cross-section was fine polished by lowering the ion beam current down to 200 pA. Subsequently, layers from the fine polished cross-section were serially milled by scanning the ion beam parallel to the surface of the cutting plane using the same ion beam current. To mill each layer, the ion beam was automatically moved closer to the surface of the cross-section by preset increments of 18.9 nm, which corresponded to the thickness of the layers. This layer thickness was verified by sectioning the reconstructed stack of images perpendicular to the original cutting plane. These reconstructed images were compared to the original SEM images and they displayed no evidence of distortions or apparent jumps in the thickness of the layers. The section thickness was also verified independently by measuring the diameter of mitochondria according to the method described by Fiala and Harris (2001a (link),b (link)). When tissue shrinkage was taken into account (see below), the mean thickness of the layers was corrected to19.9 nm.
After the removal of each slice, the milling process was paused and the freshly exposed surface was imaged with a 2-kV acceleration potential using the in-column energy selective backscattered electron detector (EsB). A 30-μm aperture was selected for imaging and the retarding potential of the EsB grid was 1500 V. The milling and imaging processes were continuously repeated and long series of images were acquired in a fully automated procedure. For this study, we obtained images of 2048 × 1536 pixels, at a resolution of 3.7 nm per pixel, thereby covering an area of 7.577 × 5.683 μm before correction for shrinkage. Under these conditions each milling/imaging cycle took approximately 4 min. Samples up to a size of a 10-cm wafer, with a height up to 4 cm, can be loaded via a load-lock and they can be completely accessed.
The three-dimensional study of the samples was carried out using a combined FIB/SEM microscope (Crossbeam® Neon40 EsB, Carl Zeiss NTS GmbH, Oberkochen, Germany). This instrument combines a high resolution field emission SEM column (Gemini® column, Carl Zeiss NTS GmbH, Oberkochen, Germany) with a focused gallium ion beam which permits material to be removed from the sample surface on a nanometer scale. Regions of the neuropil were chosen on the surface of the tissue block for 3D analysis. A protective layer of carbon was deposited on top of the area to be analyzed using an ion beam with a 30-kV acceleration potential. Using a 10-nA ion beam current, a first coarse cross-section was milled as a viewing channel for SEM observation. The exposed surface of this cross-section was fine polished by lowering the ion beam current down to 200 pA. Subsequently, layers from the fine polished cross-section were serially milled by scanning the ion beam parallel to the surface of the cutting plane using the same ion beam current. To mill each layer, the ion beam was automatically moved closer to the surface of the cross-section by preset increments of 18.9 nm, which corresponded to the thickness of the layers. This layer thickness was verified by sectioning the reconstructed stack of images perpendicular to the original cutting plane. These reconstructed images were compared to the original SEM images and they displayed no evidence of distortions or apparent jumps in the thickness of the layers. The section thickness was also verified independently by measuring the diameter of mitochondria according to the method described by Fiala and Harris (2001a (link),b (link)). When tissue shrinkage was taken into account (see below), the mean thickness of the layers was corrected to19.9 nm.
After the removal of each slice, the milling process was paused and the freshly exposed surface was imaged with a 2-kV acceleration potential using the in-column energy selective backscattered electron detector (EsB). A 30-μm aperture was selected for imaging and the retarding potential of the EsB grid was 1500 V. The milling and imaging processes were continuously repeated and long series of images were acquired in a fully automated procedure. For this study, we obtained images of 2048 × 1536 pixels, at a resolution of 3.7 nm per pixel, thereby covering an area of 7.577 × 5.683 μm before correction for shrinkage. Under these conditions each milling/imaging cycle took approximately 4 min. Samples up to a size of a 10-cm wafer, with a height up to 4 cm, can be loaded via a load-lock and they can be completely accessed.
