Biologists can now observe the workings inside our cells in full color, 3-D — and soon to be real-time. As this ScienCentral News video explains, scientists have invented a way to trick a light microscope into revealing more details than ever before seen.
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In the 17th century, a Dutch fabric merchant named Antoni van Leeuwenhoek squinted through his home-rigged single-lens microscopes to see details of lice, blood, and bees up-close. Since then, advances in microscopy have allowed us to peer into tinier and tinier worlds — tissues, cells, and organelles within the cell.
Now, scientists from the University of California, San Francisco and elsewhere have tricked the light microscope into a new way of seeing inside the cell: three-dimensional, multicolor images, closer up than ever before. The team calls its new device the Optical Microscope, eXperimental, or for short, the OMX.
To get such high resolution, the OMX uses a grating to split one laser beam into several, three of which are focused to produce a striped pattern in the sample being examined. Researcher Peter Carlton explains, "The illumination pattern interacts with the dye molecules in the sample to produce images containing high-resolution three-dimensional information." Carlton, a lead author on the team's new study in the journal Science, says an advantage of the OMX is its ease of use for biological research. The microscope captures its high-resolution multicolor images "in a very straightforward way such that a researcher with a slide that was prepared for a normal microscope can bring it to our microscope and see it twice as clearly," he explains. Currently, the OMX can snap 100 2-D pictures, or ten 3-D images, every second.
Laser light passes through diffraction gratings to illuminate samples with patterned light.
To John Sedat, a leader of the consortium that developed the OMX, the microscope is part of "the early phase of a revolution with light microscopy," a revolution that he says will bring new understanding of how all living things function. New microscopy techniques, predicts Sedat, will allow us to delve deeper into "really all aspects of developmental biology, how we develop from embryos into whole organisms, just from the aesthetics of trying to understand how we're built, or what goes wrong in disease."
Right now, the OMX achieves resolutions as low as 100 nanometers, one thousand times tinier than the width of a single hair. Sedat says that at this resolution, "You to start to really see how the nucleus is put together. How chromosomes are put together." But even closer-in details are on the horizon, according to Sedat. "We should be able to go down a lot further," he says.
Tricks of the Trade
High-resolution alone isn't what's special about the OMX, according to Carlton and Sedat, who says, "My own personal view is the real power of the new microscope is the ability to deliver on extremely fast live images." To Carlton, another advantage is "how straightforward the imaging is to perform."
Nevertheless, the OMX is part of a new generation of microscopes that have pushed past what used to be considered an insurmountable limit to light microscopy. Without the special tricks used by these new-wave microscopes, a fundamental property of light — its characteristic wavelength — imposes a limit on a light microscope's best possible resolution. (Blue light has a wavelength of about 400 nanometers, and a blue light microscope's resolution limit is about 200 nanometers. Red light has a wavelength of about 700 nanometers, and a red light microscope's resolution limit is about 350 nanometers.)
As Sedat puts it, "Resolution was limited by the physics" of light. In other words, if you peered through perfect microscope lenses to observe a pair of objects spaced by 250 nanometers, you'd see one blob, not two.
Decades ago, researchers showed that in theory, it was possible to push past the 200 nanometer limit, but that boundary breaking took years to achieve in practice. Carlton says, "It's only in the last decade or so that people have been able to build microscopes that actually do it. It's more like the sound barrier than the light speed barrier, in that it can be overcome with the right equipment."
Sedat says the OMX microscope was "put together on a shoestring budget but with no compromises. It just indicates that it is possible to fund this high-technology without huge budgets."
The University of California holds the patents to the microscope, which is being manufactured by a Seattle-based company called Applied Precision. Sedat says, "The second one is just being installed at the University of Oxford. And there are a lot of people that would love to get their hands on one, which now they can."
He adds that by rebuilding the original table-sized "breadboard" model into a practical laboratory tool, his team created a microscope "that a beginning student can learn to use this new technology in an afternoon or two."
Besides the light microscope, biologists use other types of microscopes to investigate the inner workings of our cells. Another important biology tool, the electron microscope can achieve high resolutions because it uses short-wavelength electrons to illuminate a sample. But the electron microscope is not a replacement for the light microscope. For one thing, light microscopes can collect information in color while electron microscopes cannot. Also, to use an electron microscope, researchers need to prepare their samples by freezing or other procedures that shut off movement or kill the sample.
In contrast, light microscopy does not disturb the hustle-bustle inside a cell: proteins trafficking about, DNA unwinding and replicating, virus particles budding off of membranes. The trick is to get high enough resolution so that a researcher actually can witness these events.
Sedat explains, "In the past, the resolution limit was about 250 nanometers... so a chromosome was pretty blurred. The nucleus, you could only see some aspects of the nuclear organization." With new techniques, "we can go down to approximately 100 nanometers," he says.
What's more, he says, "It's not out of the question that you might be able to see, in the very near future, 30 nanometer resolution, which is starting to knock on the door of what electron microscopy can deliver."
Along with Carlton, the lead author on the paper is Lothar Schermelleh. He and principle investigator Heinrich Leonhardt are both based at the Ludwig Maximilians University Munich. David Agard, another UCSF researcher, collaborated with Sedat on development of the OMX microscope, as well as the older DeltaVision microscope, also sold by Applied Precision.
The study was published in Science on June 6, 2008. It was funded by the Bavaria California Technology Center, the Center for NanoScience, Nanosystems Initiative Munich; Deutsche Forschungsgemeinschaft; NIH; David and Lucile Packard Foundation; NSF; Keck Laboratory for Advanced Microscopy. P.M.C., L. Shao, L.W., and P.K. have performed limited paid consulting for Applied Precision, which is planning a commercial microscope system using three-dimensional structured illumination.