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.

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.

The tissue sample is first sliced using a vibratome; the resulting block of tissue is then stained with heavy metals (Osmium, Lead, and Uranium), and then embedded in an epoxy resin (plastic). This plastic "block" is then placed into a microtome, where it is sliced into ultrathin slices of approximately 70 microns. As each slice is produced it falls into a small basin of water, and is then picked up, sequentially, on a strip of tape. The tape looks much like a film strip, with each slice lined up one after the other.

The strip of tape is cut into shorter strips, and these are in turn mounted on "wafers." The wafers are then imaged using scanning electron microscopy (SEM). The SEM produces images by scanning with a focused beam of electrons. The electrons interact with the heavy metals we stained our sample with. This produces various signals that contain information about the sample’s surface composition. The SEM produces a huge number of 2D images (it images each slice individually); these 2D images are then stacked on top of one another to form a high resolution 3D representation of the original tissue sample.

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.