Two-photon imaging gives real-time video of cells in living tissue

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.

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.

With two-photon imaging, thymus cells, dubbed beeliners, were identified moving nearly two centimetres - almost an inch - per hour, which is fast in the realm of cell movement.

Two-photon imaging is a variation on the standard technique of labelling 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 colour 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 3D image of the cells in real time. The system they use employs an infrared laser emitting short intense pulses of 920 nanometre-wavelength light.