Nonlinear (harmonic generation) optical microscopy

In recent years, light microscopy, particularly fluorescence microscopy, has been extensively useful in the study of living cells and tissues. Although it has become an important tool in biological research, both single-(Sheppard and Shotton, 1997) or twophoton (Denk et al., 1990; Cheng et al., 1998, 2001) excitation schemes require that the specimen contain either intrinsic or extrinsic fluorescent probes. These probes include fluorescent dyes, fluorescent proteins, and quantum dots and common problems include probe penetration, probe toxicity, and photobleaching\ damage (Konig, 1995; Cheng et al., 2001a). To be useful, the fluorescent probes must usually be bound to specific biostructures or molecules, either by partition of the dye into various compartments, antigen-antibody reactions, affinity or site-specific binding of dye(s), or the transgenic expression of fluorescent and luminescent proteins. In addition, the probe may interact with the ionic environment to give a specific fluorescence signature. In all these cases, the fluorescence signals are related to the chemistry of the dye and the chemistry of the interaction between the dye and the cell or tissue or the genetic expression of the probe. Therefore, the term chemical and biochemical contrast is frequently used to describe the fluorescence imaging modality.