Improved Technology for Focused-Ion-Beam Thinning of Tissue for Cryo-Electron Tomography. Application to Triad Junctions

The use of focused-ion-beam milling (FIB) to thin vitreously frozen cells for application of cryo-electron tomography is an emerging technology. However, successful application of cryo-FIB milling and tomography to frozen biological tissue has, to our knowledge, not been reported. This is because bulk tissue cannot be effectively frozen by simple plunge freezing of EM grids, but rather must be prepared by high-pressure freezing, which requires extra steps. The cryo-transfers that are required at each step of the process can cause devitrification, excessive frost contamination and mechanical damage, consequently resulting in unacceptably high failure rates. To address these problems, we have devised a protocol and have designed new devices for manipulating the specimens as they are transferred from the high-pressure freezer, to a cryo-ultramicrotome (for rough trimming), to the FIB instrument, and finally to the transmission electron microscope. Using these improved procedures we have thinned toad fish swim bladder muscle to less than 150 nm, which is necessary to achieve resolutions of 2-3 nm, and we have determined the first cryo-tomograms of vitreously frozen triad junctions. The tomograms should reveal the three-dimensional architecture of the triad with an unprecedented fidelity and level of detail. (Supported by NIH).