Researchers have combined two microscopic imaging techniques in one microscope, providing scientists with a high-resolution method of tracking single molecules in a cellular context. The development opens the door to improving our ability to visualize, in minute detail, what’s happening inside cells.
These days, scientists have the means of peering inside cells using incredibly powerful microscopes. It’s important that they’re able to do this to understand how specific biomolecules act and react. However, these tools have some drawbacks.
Take, for example, super-resolution fluorescence microscopy (SRM). It’s great for tracking single molecules, like proteins, in a cell but doesn’t show scientists what’s happening nearby. And, while cryogenic electron tomography (cryo-ET) yields high-resolution images of cells, it can’t pinpoint what individual molecules are up to.
So, researchers at the US Department of Energy’s Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory set about combining the two imaging techniques into one microscope.
“The goal is to keep the best of both worlds,” said Peter Dahlberg, lead author of the study. “You’re retaining the molecular specificity of fluorescence microscopy, so you know who’s who, and then you can put it in the context of these high-resolution structures from cryo-ET.”
Fluorescence microscopy involves tagging an individual molecule with a smaller molecule that glows when a light is shone on it. The molecule can then be tracked under an ordinary – albeit very high-resolution – optical microscope. Cryo-ET uses electron microscopes to study flash-frozen samples, like cells.
Combining the two techniques immediately raised problems the researchers needed to overcome. The first was that cells containing fluorescently labeled molecules had to be dropped onto a cryo-ET grid only 3 mm in diameter, then flash-frozen quickly enough that the water on the grid turned to glass (vitrifies). Once frozen, the cell has to stay frozen. The second problem is the size of frozen cells – they are thousands of nanometers thick – but the electrons used in cryo-CT can’t penetrate deeper than 200 nanometers.
So, the researchers developed a device called a focused ion beam milling system with an attached scanning electron microscope, or a FIB-SEM. The focused ion beam cuts away cellular material, leaving a very thin slice of frozen cell that cryo-ET can penetrate. Then, the scanning electron microscope shoots electrons at the sample to produce high-resolution images.
There was just one problem with the prototype FIB-SEM: it didn’t have an optical microscope attached, which means that the cryo-ET grid had to be moved to perform fluorescence microscopy. Luckily, there was a simple fix.
“Essentially, we just ripped apart this $1.5-million sophisticated instrument to install this integrated light microscope, and now we have a much, much better system,” Dahlberg said.
Testing the FIB-SEM in 2020, tracking proteins within bacterial cells, the researchers found it worked but realized the material the cryo-ET grid was made from was absorbing light and ruining the frozen samples. So, they made some tweaks, engineering better grids and making a better stage for the light microscope.
Now, the researchers are engineering different kinds of fluorescent labels – biosensors – to work under cryogenic conditions. The biosensors are fluorescent molecules that change their emission or excitation properties depending on the local environment, glowing one color in one environment and a different color in another.
“They can be tuned to be sensitive to pH, calcium – you name it,” said Dahlberg. “There are hundreds of environmental variables they can be tuned to. So, on top of the specific location and high-resolution structural information, you can also know was my cell healthy or sick? About to undergo cell division? At a high ATP concentration? It provides all this extra content.”
The researchers will continue to tinker with the FIB-SEM until it’s optimized and reaches its full potential.
