Development of cryo electron microscope that elucidates the structure of biomolecules such as proteins
The “Cryo Electron Microscopy”, having won the Nobel Prize in 2017, is an epoch-making analysis method enabling observation of biomolecules, such as proteins, in a ”live state”, that is, in a freeze fixation state achieved by rapidly freezing a specimen at a very low temperature. JEOL Ltd. developed a cryo electron microscope called “CRYO ARMTM” to realize this method in 2017 and put it on the market. The CRYO ARMTM allows for storing up to 12 specimens in a frozen state and removing or replacing one or more specimens as desired. It is suited for this method where a high throughput is required.
Observing proteins in a “live” state
In a human body, more than 300,000 types of proteins exist and maintain our lives by fulfilling their various functions. Each protein has a unique three-dimensional structure, and proteins having the same molecular formula exhibit completely different properties when their structures are different.
It is important to observe this three-dimensional structure in order to analyze biological macromolecules like proteins. However, a conventional X-ray crystallography requires the crystallization of proteins. Basically, proteins are difficult to be crystallized and will be crystallized in a most stable structure. So, it was not possible to observe the structure at the time when an external stimulus was given.
A solution to this problem is the cryo electron microscope. This electron microscope performs rapid freezing of a specimen by liquid nitrogen so as to encapsulate a “live” state specimen at that instance in an ice. This capability enables the observation of a projected image of molecules which are orienting in various directions. In addition, since a specimen is irradiated with an electron beam having a narrow energy width and high coherence, a high contrast image can be obtained.
When Dr. Richard Henderson, one scientist among the three who were jointly-awarded the Nobel Prize in Chemistry for developing cryo-electron microscopy, visited JEOL Ltd. , he encouraged the development and manufacturing team during his visit.
Development of the world’s first automated parking system for multiple samples
Takeshi Kaneko, Assistant Manager, a member of the development team for CRYO ARMTM. remarks,
“JEOL succeeded in realizing the automated handling of multiple samples that can load and unload 12 samples, which is, 4 samples each for three times and can also store the samples first in the world with CRYO ARMTM. Its function to store and keep the frozen sample enables various usages. After checking the stored samples, unnecessary samples can be removed, or a new sample can be loaded. It also became possible to load and observe an already-observed sample again now”.
For a cryo electron microscope, samples are frozen. Once a sample is removed from the sample stage, it begins to melt, making it difficult to reproduce the same state. But with this parking system, it is not necessary to remove the samples from the sample stage, making it possible to utilize a good sample effectively.
Naoki Hosogi, Assistant Manager, promotes sales of CRYO ARMTM outside the office, such as demonstration for users, preparation of document materials, data, and papers. He emphasizes the importance of the quality of the samples.
“Some of the biological samples such as proteins are so valuable that only one drop can be collected. So if the frozen sample is melted, it is a big issue. Samples should be handled with great attention since we know the painstaking efforts of researchers behind the samples.”
Strong interest from the pharmaceutical industry
The structural analysis of biomolecules is closely connected to drug discovery, so the pharmaceutical industry has a keen interest in the cryo-electron microscope. Hosogi presents data and findings obtained with CRYO ARM™ at conferences around the world, and interacts with researchers attending the conferences to gather feedback about the data and user hopes and expectations for the JEOL instruments.
He says "I go abroad once a month at a maximum. There is a lot of interest in our instrument. There used to be only one manufacturer. With the appearance of JEOL as a competitor, it is expected that the performance of the instrument will improve and the price will go down”.
Since the cryo electron microscope can reveal a structure of proteins as is, which was never possible before, its outcome is significant. For example, it has been elucidated for the first at how proteins react when a protein-stimulating chemical is added.
“When we receive a sample frozen by liquid nitrogen and are able to obtain the image that the customer is hoping for, the customer is extremely pleased. That makes us very happy.” says Hosogi.
Kaneko adds, "I really enjoy participating in the demos and seeing the customers’ pleased with the demo." The road to commercialization has been a long one, with continuous improvements implemented in response to the various requests from customers.
"The samples are cooled and stored under vacuum. In a hot and humid climate like Japan, however, water molecules are still present, forming a kind of frost when cooled, which adheres to the sample. We made a series of small improvements, such as devising ways to prevent water molecules from entering the sample, in order to avoid frosting even when samples are stored for a long time.”
Continuing instrument improvement, even after shipment
Kaneko was bothered by unexpected accidents after a prototype delivery to a customer, such as troubles arising from the instrument being used in an unforeseen manner, melting of the sample during observation owing to user’s inexperience in sample freezing, or thin ice films becoming broken.
Hosogi also has had some difficulties: A sample was not seen when he made a demo. In another time, a sample, frozen and sent by an overseas user, was opened at customs clearance and was ruined.
“I have delivered a few units so far. But the crucial point is whether our users can constantly acquire highly precise data or not. The only way to win the trust of the market is to steadily build a track record.” says Hosogi.
Kaneko also says that the key is after the first shipment.
“The key is to keep making improvements even after releasing the product, and how to grow it into an instrument that makes the customer happy. As a developer, a satisfied user is the best reward.”
Hosogi explains that it is his job to pursue the benefits for both the customer and the company.
"For my job, it is important to work on the customer side while having one foot positioned on technology development. My role is to inform the development side what kind of instrument is good for the user."
Market penetration of cryo electron microscopes starts from now. Kaneko says, "It may take 10 years for cryo electron microscopes to become widely adopted, but I want to improve it to be a more reasonable and user-friendly instrument, as Dr. Henderson expects."
Hosogi remarks, "We finally and only made it to the starting line. I would like more people to use it, and to make a contribution to the academic world." The impact of the cryo electron microscope may change the world.