On October 4th, the Nobel Prize in chemistry was awarded to three scientists for their contributions to the field of cryo-electron microscopy (cryo-EM), that is, using an electron microscope to examine samples held at very low temperatures. The laureates, Jacques Dubochet, Richard Henderson and Joachim Frank, each working on a different aspect of the technique, developed new methods that enabled significant progress in the use of the technique to visualize large biomolecules and multimolecular complexes, like proteins and protein assemblies. The ability to see the detailed structure of these molecular entities and observe their interactions is a critical step in understanding the relationships between structure and function. Beyond expanding our knowledge of the molecular basis of life, this ability holds great promise for the identification of new drug targets and the rational design of effective therapeutic agents.
Transmission electron microscopy and its application to imaging of biological molecules
In a transmission electron microscope (TEM) high energy electrons pass through a thin sample and form a projected image on a screen or image sensor, similar to the way a projector forms a magnified image of a frame of photographic film (Figure 1). In certain samples, TEMs routinely resolve individual atoms and can detect atomic displacements as small as a few picometers.
A number of fundamental problems have impeded the use of TEM to resolve atomic or molecular scale detail in biological materials, including: sample preparation, manipulation and imaging in the microscope, and interpretation of the images. The three Nobel laureates established major breakthroughs in these aspects of the whole process.
Biological molecules, such as proteins and other components of the cell, often have a structure that is maintained only when they are in the natural, hydrated environment of the cell. The electron microscope, on the other hand, is kept under high vacuum, complicating the goal to keep the biological molecule in its natural, hydrated environment.
Several techniques have been developed to prepare biological samples for TEM in such a way that they remain intact after inserting them into the vacuum system, but these are based on chemical techniques that replace the water that surrounds the molecules by solids, such as sugars or plastic polymers. Unfortunately, these techniques preserve only the larger scale structure of sample: most atomic and molecular scale detail is damaged beyond repair or even removed entirely by this type of sample preparation.
Jacques Dubochet approached the problem from a different angle. He started off with an aqueous solution of organic material, and tried to freeze it. Two important conditions must be fulfilled. First, the ice should not become crystalline, because diffraction from the regular lattice of the ice crystals interferes with imaging in the TEM. In addition, ice crystallization and the expansion that accompanies it cause structural changes and damage. Secondly, the frozen sample must be thin enough to be transparent to the electron beam. Dubochet managed to comply with both conditions by creating a very thin water film on a supported thin carbon film, and then freezing it very rapidly in a liquid ethane bath. Obtaining TEM images of biological molecules frozen in an amorphous ice film was a spectacular breakthrough!
Manipulation and image
Richard Henderson switched to electron microscopy in the seventies because of the limitations of biological structure determination using X-ray crystallography. For many biologically important molecules, such as membrane proteins, the required crystallization of the sample is very challenging if not impossible. A true pioneer, Henderson succeeded in solving the structure of a membrane protein (bacteriorhodopsin) with high-resolution electron microscopy. Although his method was not quite the same as current practice, he delivered the proof that electron microscopy could resolve these structures.
Frozen biological preparations, also called “cryo-samples,” put special demands on the electron microscope. During insertion and observation, the sample must remain cold enough (typically around 90 K) to maintain its glassy state. In addition, the sample must be protected as much as possible from contamination, especially by residual water in the vacuum system that can cover the sample and even form a crystalline surface layer. These requirements are especially challenging for the sample insertion and manipulation components of the microscope. In addition, the low atomic number of the carbon, oxygen and nitrogen atoms that compose most organic materials, generate very little contrast in the microscope. To make matters worse, the sample is also very sensitive to radiation damage, drastically limiting number of electrons can be used for imaging. The sample must not be unnecessarily exposed, pictures can only be made with a limited number of electrons per unit area, and the camera should be as efficient as possible. There have been major improvements in camera efficiency in recent years as a result of the transition from charge coupled device (CCD) cameras (containing a scintillator in which electrons generate light, which is subsequently detected) to complementary metal oxide semiconductor (CMOS) cameras that detect the electrons directly, without the need for a scintillator.
For decades after his breakthrough with bacteriorhodopsine, and still today, Henderson has contributed to the development of cryo-EM in many ways, ranging from theoretical reflections on contrast and radiation damage to the development of cameras and cryo components of the microscope.
The low intrinsic contrast generated by biological samples and the limits on allowable electron dose (due to radiation damage) result in images with very low signal-to-noise ratios. In a typical TEM image, even one acquired under optimal imaging conditions (see Figure 2), the molecules only appear as faint gray blobs.
Joachim Frank’s breakthrough was the development of a computational method to create high-resolution, three-dimensional (3D) reconstructions of the sample structure based on these low contrast, two-dimensional (2D) images. The technique involves the acquisition of a large number of 2D images of randomly oriented particles (typically ranging from 100,000 to as many as 1,000,000), which are then sorted into classes of different orientation. Each class is averaged to create a composite image with higher signal-to-noise ratio, contrast and resolution. The composite images of the various orientations are then combined computationally to create the final high-resolution 3D reconstruction. Somewhat paradoxically, Frank’s method to calculate a 3D reconstruction from this huge number of 2D projections is called Single Particle Analysis (SPA).
Breakthrough of cryo-EM
The development of cryo-EM was challenging and each of the three laureates contributed in a fundamental way. However, since their initial breakthroughs, it has taken another 20 years for the technique to gain broad acceptance. The selection of cryo-EM by Nature  as Method of the Year 2015 and this year’s recognition with the Nobel Prize affirm its value as a scientific technique. Much of the credit for the current broad acceptance must go to the developers of commercial systems that have made the technology accessible to a larger group of researchers. For example, Thermo Fisher Scientific launched the Vitrobot in 2002, in collaboration with Maastricht Instruments. The Vitrobot automated Dubochet’s freezing process, making it much easier to control and reproduce, and enabled more researchers to generate good samples. In addition, the company launched the first Krios a decade ago, and through four subsequent generations, the cryo-TEM has been optimized for the use of cryo specimens, focusing on ease-of-use and automation. Due to these commercial developments, it is now relatively routine for a large group of structural biologists to obtain reproducible high-resolution data with little effort. This is confirmed by the spectacular growth in the number of structures solved by TEM deposited in the Protein Data Bank  (see figure 3).
Cryo-EM’s ability to visualize large biomolecules and multimolecular complexes, like proteins and protein assemblies, allows researchers to see and understand the molecular mechanisms that constitute life itself. Medical investigators gain new insights into the mechanisms that underlie disease processes and pharmaceutical researchers design new drugs based on the structures and interactions of the drugs’ targets. The three laureates have paved the way for a technique that, on the one hand, helps us to understand the relationships between structure and function at the smallest of scales, and on the other, helps to improve the lives and health of people all over the world.
References Nature Methods Vol 13, no. 1 (Jan 2016), p1; DOI: 10.1038/nmeth. 3730  www.wwpdb.org, see www.rcsb.org/pdb/statistics/contentGrowthChart.do?content=explMethod-em&seqid=100 for the data shown in the graph