Any science student knows you have to focus a light microscope onto a sample to get a sharp image. Right? Wrong. Researchers have found a practical way to extract clear images from the parts of a sample illuminated by light above and below the focal plane. The new method may soon allow doctors to diagnose tumors without removing a piece of tissue from a patient.
Physically removing suspicious-looking cells from the body for a biopsy is necessary because conventional microscopes can only obtain a sharp image at a single depth—namely the spot where light is focused. The new method scans down through several millimeters of tissue at once and constructs a clear image of the whole region.
"We had this preconceived notion that what we imaged had to be in the focal plane," says physician and bioengineer Stephen Boppart of the University of Illinois at Urbana-Champaign, lead researcher on the project. "Now we can collect 3-D volumes of data that have the same resolution as previously available only at the focus," he says. "This might allow us to scan very large areas of tissue at very high resolution, and do that very quickly."
The secret is capturing multiple reflections from out-of-focus objects and crunching numbers to reconstruct their images, similar to techniques used in some x-ray and radar scans. Boppart says that his group has already used the method to produce images in real time. "That's really neat if they can do that," says biomedical engineer Joseph Izatt of Duke University. "That overcomes a fundamental limit in microscopy."
The technique relies on a property of laser light called coherence, meaning it is composed of electromagnetic waves all vibrating in unison. When that light reflects from a surface, the reflected light waves remain coherent but are slightly offset from each other in position, creating interference between them. The so-called optical coherence microscope, developed more than 15 years ago, reconstructs a sharp image from the interference pattern (a group of light and dark spots) emanating from the focal plane of the laser light.
Boppart says he and his colleagues observed they could reconstruct the same sort of high-resolution images from interference patterns produced above and below the focus, too. "The information is there; it just has to be processed in a different way," he says.
The incoming light waves outside of the focal plane become increasingly curved, like hands cupped around the focus. The trick, Boppart says, is to see each of the curved waves as made up of many straight waves each pointing in a different direction. As this set of straight waves reflects from a single object such as a cell, it creates its own interference pattern, which a computer program can break down into a high-resolution image, the group reports in this month's Nature Physics.
Extending the technique to tumors should require only small modifications to existing scopes inserted into the body during surgery or exams, Izatt says. "If you could do an optical biopsy through a needle or a catheter you could get the information back immediately" and potentially begin treatment then, too, he says.