Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future.
Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used "negative stains". Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low dose imaging techniques to avoiding radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM.
Taylor
Kenneth A
KA
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA. taylor@bio.fsu.edu
Glaeser
Robert M
RM
D020285:Cryoelectron Microscopy / Q000266:history* / Q000379:methods / Q000639:trends; D005615:Freezing; D049673:History, 20th Century; D049674:History, 21st Century; D013048:Specimen Handling / Q000266:history* / Q000379:methods; D014867:Water
D014867:Water