When Two Is Better Than One: Elements of Intravital Microscopy

O ver the last 20 years, many cell biological studies have moved from the single-cell level to the tissue level, and even to whole animals. This progress has been led by developments in fl uorescence microscopy that permit molecular observations from single cells within intact tissue or animals. Concurrent developments in fl uorescent probes, especially the cloning of the Green Fluorescent Protein and its use in transgenic animals, have also fueled this movement. The key instrumental technology for this work is optical sectioning microscopy; in this technique, instead of fi xing and physically sectioning a sample, the investigator obtains a 3-D dataset from an intact (and more importantly, live) specimen. The most common optical sectioning technique is confocal microscopy, where fl uorescence is created throughout the sample and a confocal pinhole is placed in front of the detector so that only the in-focus fl uorescence is recorded. For live samples, whose cells can be killed by the excitation light (via photo-toxicity, particularly of ultraviolet and blue wavelengths), confocal microscopy may not be an option. A more recently developed optical sectioning method is two-photon excitation microscopy (which also goes by the names multi-photon microscopy and nonlinear optical microscopy). As described below, two-photon excitation offers very signifi cant advantages for the high-resolution imaging of thick living samples (as deep as 1 mm). Most importantly, two-photon imaging is now ready for prime time because of instrumental advances that have made it as easy to use as any other fl uorescence microscopy technique. To understand two-photon excitation and its advantages for imaging, it is helpful to understand a little bit about fl uorescence. Fluorescence is the process of absorption and re-emission of light. Normally, a single light particle (photon) is absorbed by a fl uorescent molecule, causing an excited state, which subsequently relaxes by emitting another photon. The excitation light is typically ultraviolet, blue, or green. Any time a photon that has the correct energy to cause the excited state comes in close contact with a fl uorescent molecule, it may be absorbed. In contrast, two-photon excitation of fl uorescence depends on the simultaneous absorption of two photons (each of which contains half the energy, typically red or infrared, needed to cause the excited state). For this simultaneous absorption to happen, the photons must be so crowded that there is a good chance two photons will simultaneously be at the same place as the …