Using a tiny, spherical glass lens sandwiched between two brass plates, the 17th century Dutch microscopist Antonie van Leeuwenhoek was the first to officially describe red blood cells and sperm cells in human tissues, and observe “animalcules” — bacteria and protists — in the water of a lake.
Increasingly powerful light microscopes followed, revealing cell organelles like the nucleus and energy-producing mitochondria. But by 1873, scientists realized there was a limit to the level of detail. When light passes through a lens, the light gets spread out through diffraction. This means that two objects can’t be distinguished if they’re less than roughly 250 nanometers (250 billionths of a meter) apart — instead, they’ll appear as a blur. That put the inner workings of cell structures off limits.
Electron microscopy, which uses electron beams instead of light, offers higher resolution. But the resulting black-and-white images make it hard tell proteins apart, and the method only works on dead cells.
Now, however, optics engineers and physicists have developed sophisticated tricks to overcome the diffraction limit of light microscopes, opening up a new world of detail. These “super-resolution” light microscopy techniques can distinguish objects down to 100 nanometers and sometimes even less than 10 nanometers. Scientists attach tiny, colored fluorescent tags to individual proteins or bits of DNA, often in living cells where they can watch them in action. As a result, they are now filling in key knowledge gaps about how cells work and what goes wrong in neurological diseases and cancers, or during viral infections.
“We can really see new biology — things that we were hoping to see but hadn’t seen before,” says molecular cell biologist Lothar Schermelleh, who directs an imaging center at the University of Oxford in the United Kingdom. Here’s some of what scientists are learning in this new age of light microscopy.
Overcoming the diffraction limit
Super-resolution microscopy uses a variety of techniques to detect detail that would normally be hidden by the diffraction limit, Schermelleh explains. Single-molecule localization microscopy, for instance, takes advantage of the fact that spots on an image are easier to localize with precision when they appear in isolation rather than clustered together. Scientists label the molecules of interest with fluorescent tags designed to spontaneously emit light. As the probes twinkle on and off, computational models estimate exactly where each molecule is located — and reconstruct a high-resolution image of the sample.
Another technique, stimulated emission depletion, scans the samples with lasers that are surrounded by a second, donut-shaped ring of lasers that cancel out the fluorescent light around the area of interest, sharpening the microscope’s resolution. A third method, called structured illumination microscopy, illuminates samples with a striped pattern of light. These stripes interfere with the light emanating from the sample in ways that allow scientists to infer additional detail about the image.
The fundamentals of these techniques were developed in the early 2000s, but they’ve only recently become widespread and accessible enough for biologists to use routinely, Schermelleh says. “We now have really lots of projects that use super-resolution microscopy as a genuine tool for biological discovery,” he says, “not just for making nice images.”
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