ABSTRACT
In 1990, the publication of “Two-photon laser scanning ¦uorescence microscopy” in Science by Denk, Strickler, and Webb has ushered in an era of unprecedented growth in applying nonlinear optical principles in biomedical microscopic imaging and manipulation (Denk et al. 1990). Two-photon microscopy, as described by Denk and coworkers, is based on ¦uorescence contrast and remains the most important and widely used form of nonlinear optical microscopy in biomedicine. Today, more advanced techniques based on other nonlinear processes, such as second (Gannaway and Sheppard 1978) and third harmonic generation (Barad et al. 1997) or Raman signals (Cheng et al. 2002), are also rapidly ›nding applications in biomedicine. With the initial development of nonlinear optical microscopy in the late 1970s, there were few, if any, important biomedical applications that resulted from this discovery for over two decades. A²er the seminal publication in Science, the growth of biomedical nonlinear optical microscopy is almost exponential as measured by the publication output. Today, this growth has shown no sign of abating. ™is chapter aims to provide an overview of the principles and practices of this important class of imaging techniques for biomedical systems and will also attempt to identify the most promising future directions.
