Biologists have a new tool to track and videotape cells moving about inside living tissue.
Called two-photon laser-scanning microscopy, it has revealed, for example, the dramatic difference between the random wanderings of immature T cells and the goal-oriented, beeline movement of activated T cells.
"This is the first time anybody has quantitated four-dimensional data - spatial and time data - to get a picture of long-range cell migrations through tissue," said immunologist Ellen Robey, professor of immunology at the University of California, Berkeley. "The ability to directly visualize cells in living tissues has changed the way immunologists think about how cells explore their environment, how they signal to each other, and how they migrate."
Robey and post-doctoral colleague Colleen Witt are among a handful of researchers using two-photon imaging to obtain real-time images of cells throughout the top half-millimeter of a living organ, not just on the surface of tissue or within a slice.
"In our earlier studies (published in Science) we could see cells getting together, presumably signaling one another. In our current work, we observe cells that we believe have already gotten a signal beelining away," Robey said of her studies in the thymus, the immune system gland that weans baby T cells into activated helper, or CD4, cells and killer, or CD8, cells primed for combat with viral invaders. "We were surprised by how rapidly and directly the cells move to their final destination."
The technique could allow researchers in many fields of biology to track migrating cells, which biologists have discovered are common in many types of tissue, ranging from nerves to lymph nodes. To date, such long-range migrations have been inferred from observations of chemically fixed tissue at different stages of development.
"Two-photon imaging is going to change literally forever the way that we do biological science," said Witt, a developmental immunologist. "In the past, we'd take organs out, smush them up and basically do biochemistry in test tubes, or watch their behavior in a single layer of cells. It's an imaging revolution to be able to go into the native environment while keeping the intact organ alive and make movies of migrating cells."
With two-photon imaging, Witt and Robey identified thymus cells they dubbed beeliners moving nearly two centimeters - almost an inch - per hour, which is fast in the realm of cell movement. They think that these are cells that have received a signal committing them to be either a helper T cell - which aids other immune cells in fighting infections - or a killer T cell that seeks and destroys cells infected with virus.
On the other hand, uncommitted or immature T cells, what they call meanderers, wander slowly and apparently randomly around the outer layer, or cortex, of the thymus, perhaps in search of that life-altering signal.
Robey hopes to use two-photon imaging to investigate the signals responsible for changing these meanderers into purposeful beeliners that immediately leave the cortex for the interior medulla of the thymus.
"We're now at the point with this technology that we can begin to look at the movement of signaling molecules within the cells," she said.
Robey, with another colleague, Philippe Bousso, last year published a review in the journal Immunity describing the contributions two-photo imaging has made to the field. Robey and Witt publish their current study in the May 3 issue of the Public Library Of Science-Biology.
Two-photon imaging is a variation on the standard technique of labeling cells with fluorescent dye and then hitting them with a laser that makes the dye glow and the cells light up. A certain energy or color of laser light is needed to make the dye, in this case green fluorescent protein, glow. But high-frequency, short-wavelength visible light, like green, doesn't penetrate tissue as deeply as longer, redder wavelengths.
The idea behind two-photon imaging is that if you hit a dye molecule in a short period of time with two photons of light, each photon half the energy needed to excite it, the dye can absorb them together and then fluoresce. The less energetic, long-wavelength photons will go deeper into the tissue, cause less damage and scatter less, Robey said, essentially illuminating slices through the tissue that can be sharply imaged and stacked to produce a 3-D image of the cells in real time. The system they use employs an infrared laser emitting short intense pulses of 920 nanometer-wavelength light.
In the thymus, it's possible to view cells 400 microns inside the cortex, which is about 4/10 of a millimeter or more than a hundredth of an inch deep. In the current study, Witt limited her viewing to about 200 microns, though she says in some tissues less dense than the thymus, light could penetrate nearly a millimeter - deep enough to probe cell activity in most tissues.