Xiaowei Zhuang, an HHMI investigator at Harvard University, makes movies of single fluorescently tagged influenza viruses invading cells. Others had used electron microscopes to spot influenza virus in membrane-bound compartments called coated vesicles and endosomes, indicating that cells engulfed the viruses in a process called receptor-mediated endocytosis. But no one knew whether the virus homed in on existing pits in the membrane or induced the cell to create new ones.

Zhuang's movies showed a red virus surrounded by a green coat of clathrin molecules, which coat membrane pits. The pits then bud off to become coated vesicles. "We actually saw pits grow right outside a virus," she says. That means the virus most likely persuades the cell to take it up by receptor-mediated endocytosis—a result she confirmed by statistical analyses. Until Zhuang made her movies, no one knew influenza viruses used this trick to invade cells. Drugs that target key parts of the process could one day help block viral infection.

Svoboda and other researchers use two-photon excitation microscopy to peer into opaque tissues like the brain—an ability akin to Superman's x-ray vision. Optically, brain tissue resembles milk, he says. The interface between fat and water "acts like little mirrors" that scatter light and make the substance appear white, which makes it impossibly murky under older fluorescent scopes.

In two-photon excitation microscopy, however, a new type of laser emits bright, synchronized pulses of infrared light that are focused on tiny volumes in the cell. Tagged molecules fluoresce only when they simultaneously absorb two such photons. Outside of the zone of focus, that's rare, so the method dramatically reduces background. Recently, Svoboda's team watched single nerve endings fire by combining the method with a second fluorescence method called fluorescence resonance energy transfer (FRET).

FRET works like this: One fluorescent dye emits light of a specific color that excites a nearby dye to glow a different color. By tagging one protein with the donor dye and a second protein with the acceptor dye, scientists can see when and where the two proteins interact. Svoboda chose proteins that would come together only when an enzyme called Ras was activated. It turned out that activation of single synapses in the hippocampus activates Ras, which then stimulates the synapse to reshape itself—a phenomenon that underlies learning.

Recent advances in fluorescence microscopy occurred only because microscope designers focused on developing and adapting new technologies. To see GFP and its cousins, for example, they built new lenses from materials that allow for brighter samples and greater contrast, created thin-film interference filters that transmit only a specific color of light, and replaced film cameras with low-noise CCD cameras to record very dim fluorescence. Two-photon excitation microscopy required new microscope objectives that were transparent to infrared light, and mode-locked pulsed lasers, developed in the 1980s, to create very short, very bright pulses of photons.

Karel Svoboda and other researchers use two-photon excitation microscopy to peer into opaque tissues like the brain—an ability akin to Superman's x-ray vision.

The past few years have seen an explosion of new microscopy methods, which sometimes read like alphabet soup: two-photon fluorescence correlation microscopy (TPFCM), which helped scientists trace drug transport in tumors; three-dimensional live-cell microscopy, which helped identify never-before-seen thread like transport lines between live cells; the GRIN lens, a needle-shaped, insertable lens that can create microscopic images several centimeters deep in the brain.

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