Good sample preparation is the key to getting the best from cryo-electron microscopy

The images produced with a cryo-electron microscope can only be as good as the sample that goes into it. That's why Jiro Usukura, at the Structural Biology Research Center, Nagoya University, says good sample preparation technique is essential.

"The effort of producing high-resolution cryo-EM images is wasted if they are compromised by poor sample preparation," Usukura says. He advises that a good sample for cryo-EM is, most importantly, small and thin.

With cryo-EM, the sample is frozen rapidly, maintained at approximately -180^oC and observed directly in its frozen state without any other processing that could affect the molecular structure. Biological samples for conventional electron microscopes are often negatively stained or embedded into resin to produce thin sections. These methods can adversely affect the sample, dehydrating it through exposure to air.

The process for cryo-EM prevents dehydration of the sample and allows it to be viewed at the resolution of an electron microscope in a pristine state.

Professor Emeritus Usukura, in collaboration with researchers from Japan Women's University and Hitachi High-Technologies Corporation have developed new cryo-EM technology to produce transmission and surface images simultaneously in the frozen state.

“Biological samples are very fragile. If water becomes crystalized ice on freezing, fine structures are easily destroyed. Therefore, the samples must be frozen rapidly so that the water transforms into amorphous or vitreous ice. The sample must be thin and small to enable rapid freezing,” explains Usukura.

His team developed a cryo-transfer holder that allows transfer of a frozen sample into the microscope while maintaining the temperature at -180^oC, reducing the risk of crystalized ice forming.

This holder has a further benefit in the cryo-EM process. During observation in a vacuum, irradiation by the electron beam causes the ice in the sample to sublimate, producing water vapour. The sample can then be contaminated and obscured, as the vapour recrystallizes on its surface. Usukura explains that the cryo-transfer holder works in tandem with an anti-contamination trap, also developed by the team, to draw the water vapour away from the sample, resulting in higher quality images.

Cryo-electron microscopy has become a popular route for researchers to view and study biological and chemical structures such as viruses, molecules, and cells. It allows direct internal or external images, depending on the equipment used.

It’s no wonder that in 2017, the Nobel Prize in Chemistry was awarded to three researchers who had spent decades advancing and developing cryo-EM: Joachim Frank, Jacques Dubochet, and Richard Henderson.

It is important to remember that all preparation methods are developed for a specific purpose, says Usukura. It is vital to select a preparation method suitable for an image’s intended use. For example, a process known as unroofing can be used to view the membrane cytoskeleton inside the cell. Unroofing involves the mechanical removal of the cell membrane by ultrasonic pulses. Usukura and the team refined the process for their purposes by gently washing away the soluble components in the cell. They developed equipment specifically to produce more delicately unroofed samples. Using this refined unroofing process, and their new equipment, the team were able to produce three-dimensional images of the clathrin, caveoli and actin filaments interacting within the membrane — impossible by using other techniques.

Usukura says cryo-EM will become easier to use and more popular in the near future. The technology is already being used in other fields, such as materials science, and he anticipates strong demand for cryo-EM in areas such as cosmetics, pharmaceuticals, foods and soft materials.

Indeed, this equipment enabled the team to produce clear cryo-EM images of nano-bubbles formed in water. These are gas bubbles that are approximately 50-100nm in diameter, that are a current focus for research by materials scientists.

“Good preparation and cryo-EM will allow the scientist to observe materials that weren’t possible with conventional electron microscopy,” Usukura says.