Abstract
Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm2 (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm2 (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of ∼100 µm2 and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (∼45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.
Highlights
Serial thin sections of,100 nm thickness have been used to visualize and reconstruct cellular and subcellular structures in the three-dimensional (3D) context from a wide variety of biological systems
Results Transmission-mode scanning electron microscopy (tSEM) Image Quality is Comparable to transmission electron microscope (TEM) Images The new tSEM system accommodates specimens prepared in the same manner as for TEM
The autofocus routine in tSEM repeatedly scans a small area in the center of the imaging field, which leads to greater brightening of the focus area compared to the rest of scan area (Fig. 1F)
Summary
Serial thin sections of ,100 nm thickness have been used to visualize and reconstruct cellular and subcellular structures in the three-dimensional (3D) context from a wide variety of biological systems. Our laboratory and others have used ssEM to understand how the structure of synapses and neuropil is modified by experience and in models of learning and memory [17,18,19] or under pathological conditions [20,21,22,23,24,25,26] The results from these studies have provided fundamental insights into the anatomical substrates for changes in information processing and behavioral output. Both normal and pathological changes in neuronal morphology can involve subcellular structures such as, polyribosomes, microtubules, endosomes, dense core vesicles, and smooth endoplasmic reticulum, that require ssEM at nanoscale lateral resolution (,2 nm per pixel in x–y) to be reliably identifiable. Microtubules and other small organelles have been detected at lower image resolutions using other ssEM techniques (e.g., [27,28]), our experience is that reliable identification and quantification becomes difficult at lower resolutions [17,19,29,30]
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