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TechStream looks at new technologies being developed at Lawrence Berkeley Lab. If you’re interested in knowing what tomorrow’s technology will look like, then check back here frequently.

Taking the Cryo-Plunge:Flash-freezing in the field

When Berkeley Lab scientists descend into one of the most toxic places on Earth to find out what’s alive in the poisonous pink slime of California’s Iron Mountain Mine, one of the newest pieces of equipment they carry is a bird-like contraption dubbed the Portable Cryo-Plunger.

No, it’s not plumber’s helper for unclogging frozen drains.

Birgit Luef (left) and Luis R. Comolli (right) prepare cryogenic TEM samples using headlamp lights inside Iron Mountain Mine, near Redding, CA.

Birgit Luef (left) and Luis R. Comolli (right) prepare cryogenic TEM samples using headlamp lights inside Iron Mountain Mine, near Redding, CA.

It is a simple yet ingenious device for flash-freezing microbes destined for a look in an electron microscope.  The sole job of the Cryo-Plunger is to do exactly what the name implies: in a split second, it drives a tiny specimen into a bath of liquid ethane (-183 °C). It freezes the suspensions and films of microbes within an extremely thin slab of “vitreous ice” — water frozen so quickly no crystals form — as if it were encased in glass. I recently paid a visit to the Berkeley Lab inventors of the portable Cryo-Plunger, and quickly discovered how this unassuming piece of portable equipment could transform the way environmental microbiology is done today.

It turns out that freezing your samples in vitreous ice is absolutely essential if you want to study them in a Cryogenic Transmission Electron Microscope (CryoTEM). These are expensive, incredibly sophisticated instruments that use electron beams instead of light and can resolve images 1,000 times smaller than the most powerful optical microscopes. The ideal way to flash-freeze biological samples is to use an automated laboratory tool, such as a Vitrobot. But hauling an $80,000 robotic flash-freezer into an acid-laced copper mine is not just impractical. It would be crazy. A Berkeley Lab team of electron microscopists led by Luis R. Comolli knew there had to be a better way: The Portable Cryo-Plunger was born, and a roadblock to the study of intact microorganisms and their interactions in the wild was lifted.

Lightweight, fully mechanical, and easily strapped to a backpack, the device has been tried and tested for nearly two years. Machined from aluminum tubing and standing 28-inches tall, the spring-loaded gadget might be mistaken for the latest tool for uncorking a bottle of wine. Instead, it is opening up new possibilities – giving environmental field microbiologists for the first time full access to the extraordinary power of electron microscopy.

Comolli and Birgit Luef, a UC Berkeley postdoc, have taken the Cryo-Plunger into the abandoned Iron Mountain Mine, near Redding, California, to flash-freeze samples of extremophile bacteria. “You come out of there smelling like a car battery,” Luef said. “Three showers later, it’s still with you.”

Luef demonstrating the Cryo-Plunger. Set up on a camera tripod, it weighs only 4 kg. It is designed for ease of operation in the field.

Luef demonstrating the Cryo-Plunger. Set up on a camera tripod, it weighs only 4 kg. It is designed for ease of operation in the field.

Luef’s microscopy work complements that of her mentor, Jillian Banfield. The acclaimed Berkeley Lab scientist and UC Berkeley professor has used gene scans to discover remarkable new species of microorganisms that thrive in the highly acidic runoff of the mine — a federal Superfund site. With CryoTEM, detailed images of these novel organisms can be correlated with the genomics. Thanks to the Cryo-Plunger, samples from a variety of remote or extreme environments, such as mines, volcanic craters, and hot springs, now can be preserved and brought to the lab for CryoTEM study. “The sample preparation method is critical,” Banfield told me. “To my knowledge, no other research teams in the world are doing this.”

For the study of microorganisms, few pieces of laboratory equipment are more complex than CryoTEMs. They reveal the structures not only of bacteria and viruses, but even of the molecular machinery that operates inside them. Without flash-freezing, however, the benefits of such precision imaging are lost on samples taken from remote sites. Like a flash camera, the freezing process also captures these microbes in the instant of their interactions with their natural environment, at the moment they absorb nutrients, respire, replicate, or simply coexist and interact with other organisms  in an ecosystem — a moment impossible to recreate with cultured bugs back at the lab. In fact, only a small minority of microbes can be kept alive and cultured for flash-freezing outside their natural environs. As for the rest, the delicate traits and features of microorganisms can be destroyed when they are collected, stored, and transported. As such, CryoTEM has been largely out-of-reach for environmental microbiologists.

The Cryo-Plunger is changing this. Comolli, who works with a state-of-the-art CryoTEM at Berkeley Lab, showed me how the plunger works. “We designed this to minimize the tasks done by the user,” he said.  In the simplest terms, it’s a spring-powered piston. It grips a sample, and plunges it into a bath of liquid ethane, which has heat-sucking properties perfectly suited for flash freezing. The business end of this device wields a standard laboratory tweezers that grips a tiny ring (TEM grid) at the tip. A thin film of water loaded with living specimens is deposited onto the TEM grid. Excess liquid sample is wicked off with a piece of filter paper. The trigger at the top of the plunger is pressed, and in an instant the tweezers-tip and grid are immersed in a thimbleful of very, very cold liquid. Plexiglass splatter guards keep the process safe. Conveniently, the thimble rests in a cup-sized vat of liquid nitrogen, which not only keeps the liquid ethane cold, but serves as a freezer to store up to 16 vitreous-ice samples. Eventually, they’ll be transferred into a thermos of more liquid nitrogen for the journey back to the lab.

Microbial ecologist Luef has used the Cryo-Plunger to take more than 500 samples from another remote Superfund site, in Rifle, Colorado. Uranium-contaminated groundwater is being treated there with acetate, which stimulates bacteria that convert soluble uranium into an insoluble form. This chemical process, the microbial “reduction” of metals, is of great interest to researchers looking for better ways to clean-up toxic sites. Under the Cryo-TEM, her flash-frozen samples showed a species of Geobacter covered with aggregates of nano-sized particles of iron, which further tests will show were either waste-products of the bacterium, or its source of energy. “It could be as if the bacterium carries around its own oxygen tanks, only this bug is breathing iron,” said Luef.

(Three-dimensional Cryo-TEM reconstruction of a bacterium (probably Geobacter) preserved using the Cryo-Plunger at a Superfund site in Rifle, CO. The bacterium is covered with nano-aggregates of iron attached to the cell surface.)

Until the Portable Cryo-Plunger came along, field microbiologists typically would have to settle for electron microscope images of bugs that were already compromised: the bacterial equivalent beef jerky. The more remote and extreme the environment, the harder it is to preserve a useful sample. As a result, “You simply couldn’t do it with extremophiles,” said Roseann Csencsits, manager of Berkeley Lab’s TEM facility in LBNL’s building 1. With the Portable Cryo-Plunger in hand, however, a new chapter in microbiology is being written. As Comolli put it, “We have seen stuff that no one has seen before.”

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