Serial block-face scanning electron microscopy (SBFSEM)
Serial block-face scanning electron microscopy (SBFSEM) is a method for generating high resolution 3D images from biological samples. It was created at the Max Planck Institute in Germany, specifically for the purpose of imaging neurons.
How EyeWire uses SBFSEM
The images you see on EyeWire come to us from our collaborators Kevin Briggman, Moritz Helmstaedter, and Winfried Denk at the Max Planck Institute in Germany, and the dataset is called e2198.
Before any imaging could be done, the sample was stained with heavy metals. When the scanning electron microscope's electrons collided with the heavy metals in the sample, they would bounce off and they would be collected by a detector. These electrons that bounce off the sample are known as backscattered electrons. After being placed inside the chamber of the microscope, the surface of the sample is imaged. Because the Scanning Electron Microscope uses a tightly focused beam of electrons, the sample needs to be scanned in a certain pattern. The Scanning Electron Microscope will move across a line, scanning it one piece at a time, and then it will move onto the next line. This is known as raster-scanning.
After the entire surface of the sample has been imaged, an ultramicrotome slices off the surface of the sample and the underlying surface is then imaged in the same way. The images from the scans of each successive layer of the sample were combined to form a 3D dataset.
Think of the the stack of 2D images like a flip book.The way flip books work is that from one page to another there is a small change in the drawing, and several small changes in a row create the action in the flip book. The 2D image you see in EyeWire is like one single page from a flip book. The 2D is static, but when you scroll through the slices you can see the change from one slice of retina (or page of a flip book) to another. The idea is that if you follow the shape of one neuron from one 2D slice to the next, coloring each piece as you go, you can eventually discern the shape of the neuron in the 3D. Each piece you color in the 2D builds upon the previous one. It's like stacking blocks, each layer of blocks is flat, but as you continue stacking the blocks on top of each other you eventually get a 3D shape.
For a less technical explanation you can view the article on the blog.
- Denk W, Horstmann H (2004) Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure. PLoS Biol 2(11): e329. doi:10.1371/journal.pbio.0020329