Prática de Shadowing: Introduction to focused ion beam scanning electron microscopy (FIB-SEM) - Aprenda a falar inglês com o YouTube

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Hi, I'm Kedar Narayan, a group leader at the Center for Molecular Microscopy, or CMM, at the National Cancer Institute and Frederick National Laboratory for Cancer Research.
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Hi, I'm Kedar Narayan, a group leader at the Center for Molecular Microscopy, or CMM, at the National Cancer Institute and Frederick National Laboratory for Cancer Research.
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Imaging three-dimensional objects in 2D can be limiting, yielding an incomplete or even a misleading picture.
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And so, at the CMM, we apply cutting-edge electron microscopy, or EM, technologies to image biological samples at the highest possible resolutions.
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My group specializes in the imaging of cells and tissues, that is, larger volumes, in 3D and at nanoscale resolutions.
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In this short video, I'm going to give you a flavor of an imaging technique that we employ in our lab, Focused Ion Beam Scanning Electron Microscopy, or FibSem.
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FibSem, as you will see, is a powerful 3D-EM imaging approach, and in this specific example, we highlight a correlative light microscopy and FibSem, or CLEM-FibSem workflow.
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CLEM-FibSem combines the advantages of light and electron microscopy to generate image reconstructions of targeted features of interest in 3D at resolutions of tens of nanometers.
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So here you have a group of cells of which a subset is fluorescently labeled.
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These cells are grown on a gridded cover slip, a glass substrate on which an alphanumeric code has been etched.
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You can now image these cells either live or after fixation using fluorescence microscopy and also record their x-y coordinates using the gridded pattern.
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Once this step is completed, the cells can be fixed, stained, dehydrated, and resin-embedded in situ.
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When the gridded coverslip is removed, the alphanumeric pattern is transferred to the resin surface in relief, with the cells embedded just beneath that surface.
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In other words, you now know exactly where to find your cells of interest.
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At this point, the cell sample is transferred to the FibSem instrument.
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This Martian looking surface is a close up of the resin where you expect to find your targeted cell.
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The resin embedded cell is protected by a patterned platinum and carbon pad deposited by the focused ion beam or fib, which appears as a rapidly moving blue beam in this movie.
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Remarkably, the same focused ion beam that deposits the platinum and carbon pad can now be used to mill a trench in front of the targeted cell.
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The tightly controlled fib inches towards the cell, ablating away the resin, and eventually reveals the cell itself in cross-section.
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The scanning electron imaging beam, shown here as a yellow band, now rasters over the polished resin surface, and a detector records a high-resolution backscatter electron signal.
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This process is repeated over and over.
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As the fib moves forward and mills away a few more nanometers of the resin, the SEM images the newly exposed section of the cell to generate the next image, and so on and so on.
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Typically, we generate SEM images at 3 to 5 nanometer pixel size in the imaging plane, and 3 to 15 nanometer fib step size, meaning that this automated loop is often repeated several thousand times to generate a highly information-rich ultra-structural image stack covering entire mammalian cells.
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These images are then registered using the notch marks in the pad and converted to an isotropic image volume that allows you to visualize architectural features throughout the bulk of the cell sample.
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The final step is segmentation.
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Here we show the extraction and rendering of the plasma membrane of the cell.
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Fibsem imaging reveals that the membrane extensions in the cell that looked like spaghetti in 2D cross-section are actually veils when visualized in three dimensions.
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FibSem imaging can be applied to a wide variety of systems to confirm observations, generate hypotheses, and most fun of all, make unexpected discoveries in cell biology.
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If you'd like to know more, you can contact me, Kedhar Narayan, at the Center for Molecular Microscopy at the National Cancer Institute and Frederick National Laboratory for Cancer Research.
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You can also visit cmm.nci.nih.gov.

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