Confocal microscopy, however, can penetrate only around 80 μm into a tissue, and at the wavelengths typically used for excitation, the source light will soon bleach the fluorescent label and damage the specimen. This means that the technique is not suitable for imaging a live specimen over a period of time sufficient, for example, to track the movements of cells in a tissue. The more recently developed technique of twophoton scanning fluorescence microscopy overcomes some of these limitations. In this technology, ultrashort pulses of laser light of much longer wavelength (and thus with photons of lower energy) are used for excitation, and two of these lower-energy photons arriving nearly simultaneously are required to excite the fluorophore. Excitation will therefore occur in only a very small region at the focus of the microscope, where the beam of light is most intense, and so fluorescence emission will be restricted to the plane of focus, producing a sharp, high-contrast image. The longer-wavelength light (typically in the near infrared) is also less damaging to living tissue than the blue and ultraviolet wavelengths typically used in confocal microscopy, and so imaging can be carried out over a longer period. More of the emitted light is collected than in confocal microscopy, and because single photons scattering within the tissue cannot cause fluorescence and consequent background haze, imaging to greater depths (several hundred micrometers) is possible. Like confocal microscopy, two-photon microscopy produces thin optical sections from which a three-dimensional image can be built up.
To track the movements of molecules or cells over time, confocal or two-photon microscopy is combined with timelapse video imaging using sensitive digital cameras. In immunology, time-lapse two-photon fluorescence imaging has been particularly valuable for tracking the movements of individual T cells and B cells expressing fluorescent proteins in intact lymphoid organs and observing where they interact (see Chapter 10).
A-11 Immunoelectron microscopy.
Antibodies can be used to detect the intracellular location of structures or particular proteins at high resolution by electron microscopy, a technique known as immunoelectron microscopy. Antibodies against the required antigen are labeled with gold particles and then applied to ultrathin sections, which are then examined in the transmission electron microscope. Antibodies labeled with gold particles of different diameters enable two or more proteins to be studied simultaneously (see Fig. 6.12). The difficulty with this technique is in staining the ultrathin section adequately, because few molecules of antigen are present in each section.
A-12 Immunohistochemistry.