title: Automated in-chamber specimen coating for serial block-face electron microscopy.
abstract: When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally ('sample charging'). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block-face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1-2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal-to-noise ratio (SNR) caused by the film is smaller than that caused by the widely used low-vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non-conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.
authors: Department of Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg, Germany.
authors: B
authors: B
authors: Titze
authors: Department of Biomedical Optics, Max Planck Institute for Medical Research, Heidelberg, Germany.
authors: W
authors: W
authors: Denk
mesh_terms: D000818:Animals; D001479:Basal Ganglia / Q000648:ultrastructure; D001921:Brain / Q000648:ultrastructure; D046369:Finches; D005404:Fixatives; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008867:Microtomy; D009993:Osmium Tetroxide; D011817:Rabbits; D012160:Retina / Q000648:ultrastructure; D059629:Signal-To-Noise Ratio; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D016610:Tissue Embedding
chemical_list: D005404:Fixatives; D009993:Osmium Tetroxide