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.
Witt pointed out that obtaining a movie of cell movement is just the beginning. The human eye and brain can't pick out patterns of movement easily, so statistical techniques are needed to identify cells with different patterns of movement.
As an immunologist, Robey focuses on the lives of T cells produced in the thymus and distributed via the bloodstream to the lymph nodes, whence they move into the body's tissues. Her first use of two-photon imaging three years ago surprised her and many immunologists because it showed that thymocytes or immature T cells were highly mobile, traveling thousands of microns in an hour as they explore the thymus.
The new experiments, conducted primarily by Witt,
Source:University of California - Berkeley