Two-photon tissue imaging: seeing the immune system in a fresh light

Two main techniques have been used to analyse the immune system. In vivo experiments examine cell populations in their natural environment, but they cannot provide information about individual cells. In vitro experiments determine subcellular and molecular details, but they cannot replicate adequately the physiological environment. Therefore, new techniques, involving fluorescent labels, are required to analyse single cells in intact tissues in real time. Three main problems limit optical resolution when attempting to visualize fluorescently labelled cells in living tissues. First, the high-numerical-aperture lenses that are required have a narrow depth of field, so that fluorescent label from above and below the focus obscures the image. Second, biological tissues scatter light, which reduces image contrast. Third, the intense excitation light causes photodamage and photobleaching. A partial solution is provided by confocal microscopy, which provides a sharp 'optical section' at a given depth by rejecting out-of-focus fluorescence from above or below the focal plane. But, light scattering limits the depth of imaging and the specimen is susceptible to photodamage. Two-photon microscopy has the advantages of greater imaging depth (owing to reduced light scattering of the long wavelengths that are used for excitation) and minimal photobleaching (as excitation is confined to the focal plane). Two-photon excitation involves the simultaneous absorption by a fluorophore of energy from two photons, each of which contributes one half of the total energy required to induce fluorescence. The probability of two-photon excitation falls off rapidly from the focal point. Pulsed lasers (such as the titanium-sapphire laser) are required to provide the necessary high photon density at the focal spot without vaporizing the specimen. The peak power during a pulse is extremely high, but the average laser power is relatively low. The choice of objective lens is also important to maximize the numerical aperture while maintaining imaging depth. 'Dipping' water-immersion objectives provide the best compromise. Two-photon microscopy allows imaging in as many as six dimensions (three spatial dimensions, time, intensity and wavelength of more than one probe). This creates problems in terms of storage, analysis and representation of the data that will require new solutions, such as the use of virtual-reality technology. As an example, two-photon techniques have been used to examine the movement of T cells in lymph nodes and their response to antigen. These studies have indicated that both the single-encounter model and the serial-encounter model of antigen presentation might be relevant physiologically.