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Each ribbon consists of roughly 15 sections, so the number of samples that can be acquired at once is approximately 50.ī: Collecting sections using the iron ring.Ĭ: Adhesion of sections. 2F).įinally, the sections are observed by SEM system (Hitachi SU3500) equipped with a thermionic electron gun and a highly sensitive semi-annular semiconductor backscattered electron (BSE) detector at an acceleratingĪ.
#Three disadvantages of electron microscope serial
To avoid this problem, the serial sections are coated with platinum/palladium with a thickness of several nanometers (Fig. If the glass slides with the sections are subjected to SEM, this may result in the occurrence of artifacts due to charging. Carbon tape is then used to mount the slide on the SEM sample stub (Fig. To mount the glass slide with the sections on the aluminum SEM sample stub, a diamond pen is used to scribe a line on a glass slide (region with dimensions of approximately 1 cm×1 cm), and the slide is then carefully broken by hand. Here, the sections are stained with a 1% aqueous solution of uranyl acetate for 10min, rinsed with distilled water, and then stained with Reynolds lead solution for 5min (Fig. For this reason, the sections can be stained with heavy metals. In contrast to FIB/SEM and SBF/SEM, serial-section SEM allows the sections to be immobilized on the glass The required number of serial sections for 3D reconstruction is obtained by repeating steps 2-5. The glass slide with the sections is placed on a hot plate, which has been preheated at 60☌ for 30min (Fig. The ribbons are transferred from the ring to a glass slide.įig.1 Flowchart showing sample preparation procedure for serial-section SEM. The serial-section ribbons are picked up using the iron ring (Fig. The process of ribbon creation and separation is repeated to produce four aligned ribbons, resulting in a total of 40-50 sections (Fig. In our case, a ribbon containing 10-15 serial sections is cut using the diamond knife, and then carefully separated from the knife using an eyelash. The length of the serial-section ribbons is chosen to ensure that they can be picked up using an iron ring with an inner diameter of 7 mm. Ultrathin serial sections (100 nm thick) are cut using an ultramicrotome with a diamond knife.