![]() ![]() ![]() aimed to create a technique to image a complete fly brain, with gaps of just 8 nanometres between each image in a stack, within a reasonable timeframe. The current technical challenge is to create a system that can, for example, successfully image and analyse all the connections between the more than 100 thousand cells that make up the brain of a fruit fly – a common model organism in neurobiology. FIB-SEM would allow faster, more accurate and detailed studies of connections between brain cells, and other elaborate biological systems, if the technology could be made faster and more reliable over months of continuous operation. Unfortunately, FIB-SEM devices are currently slow and can only run for a short time, leading to a lack of continuity in the stack of images. The new surface can then be imaged and so on, through the whole sample. This approach works by creating an image of the surface layer of a sample, which is then stripped away using a beam of charged particles to reveal the layer beneath. One approach for examining particularly fine details, such as the connections between nerve cells in the brain, is called focused ion beam scanning electron microscopy (or FIB-SEM for short). ![]() These images are usually built up from many two-dimensional images stacked on top of each other. Precise three-dimensional imaging can help make sense of microscopic details in biology. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology. Even higher resolution can be achieved on smaller volumes. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 10 6 µm 3. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. ![]()
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