Bringing color to electron microscope images is a tricky problem. It could plausibly be said that color doesn’t exist at that scale, because the things imaged by an electron microscope are smaller than the wavelength of visible light. But that hasn’t stopped scientists from trying, or at least developing techniques to approximate it.
The latest, described in an article in Cell by scientists from the University of California, San Diego, attaches artificial color to biological structures, which could help us better understand the structures and functions within cells. They’re the first to use this method on organic material, matching up to three colors and making, in one example, a Golgi region appear green and a plasma membrane red.
“It adds a lot of additional information to conventional electron microscopy,” says Stephen Adams, lead author of the paper. “We hope it will be a general technique that people will use for this very high resolution mapping of any molecule, really, that they want to.”
As technologies like this drive up the resolution of images, it could allow scientists to peek inside the cells themselves, and identify the bodies within them in greater detail. Under a traditional, light-based microscope, it’s impossible to image something smaller than the wavelength of light that the microscope uses, which is around 250 nanometers, explains Brian Mitchell, an associate professor of cell and molecular biology at Northwestern University. “That’s a pretty big area, so if you’re trying to say that this really important protein you’ve found is on the inside of a membrane or on the outside of a membrane, it’s really hard to say that when you can’t get below that 250 nm resolution,” he says.
Meanwhile, the black and white images generated by an electron microscope have a similar problem: While the resolution the scope provides is great, it can be hard to distinguish between different cellular structures on a gray scale.
The technique Adams and company used is sort of a combination of light microscopy, which bounces light off of objects, and electron microscopy, which bounces electrons off of objects. First, they use a light microscope-generated image to identify the structures they want to highlight. They introduce a small amount of rare earth metal, and overlay the structure with it. Then they subject it to an electron microscope.
When the microscope fires electrons at the tissue, some go right through, and others hit thicker or heavier materials and bounce back, sort of like an X-ray. A few strike the rare earth metal, and displace an electron there, causing it to fly out; along with comes a little bit of energy, distinct to the particular metal used, and this is what their microscope is measuring. The technique is called electron energy loss spectroscopy.
Adams has imaged cell structures like the Golgi complex, proteins on the plasma membrane, and even proteins at the synapses in the brain. “For many biological experiments, it’s useful to have that very high magnification for, really seeing where these proteins are, or where this particular molecule is in the cell, and what it’s doing,” he says. “It often gives you an idea of what the function is.”
This isn’t just academic, points out Mitchell. Knowing what’s going on inside a cell can be useful in the diagnosis and treatment of disease.
“If you have a protein that, say, localizes to some cellular substructure … and maybe in that disease situation the protein doesn’t go to where it’s supposed to go,” says Mitchell. “By looking at the localization of the protein, you say, ‘hey, this protein is not going where it’s supposed to, that’s probably what’s underlying the mechanism of why the cell’s not functioning the way it’s supposed to, and could underlie why this disease does what it does.’”
The Cell article is not the only attempt to provide color imagery from electron microscopes. One other is correlative light electron microscopy, which tags cell structures in a light microscope image with fluorescent molecules to locate them, then uses an electron microscope to image them, and overlays the two images. Another is immunogold labeling, which binds gold particles to antibodies, and those then appear in an electron microscope image because of the density of the gold. But each has its own problem: the former necessitates two different images, from different microscopes, reducing precision; and the latter can give unclear staining.
The paper was the last to bear the name of Roger Tsien, a Nobel prize-winning chemist who died in August. Tsien was best known for using a fluorescent protein from jellyfish to illuminate cellular structures.
“[This paper] was the culmination of almost 15 years of work, so I think it’s another legacy that he’s left,” says Adams. “That’s the hope, that it’s going to lead forward to new ideas and new ways of improving the electron microscope and its usefulness.”