About 60 years later, the rst multiphoton excitation images (using two-photon excitation) of biological specimens were demonstrated (Denk et al. 1990).

According to the development of laser techniques, laser scanning microscopy (LSM), which uses a laser as an excitation light source for uorescence microscopy, has also been developed. In an LSM system, focused excitation laser light scans specimens sequentially point-by-point, line-by-line, or taking multiple points at once. Light emitted by uorescence passes through some optical lters and is detected by photomultiplier tubes (PMTs) or avalanche photodiodes. Finally, pixel information is assembled into one image by a computer. In particular, confocal LSM (CLSM) is the most popular methodology; this idea was rst described in a patent application by Minsky in 1957 (Minsky 1988). CLSM utilizes the pinhole in front of the detector not to detect uorescent signals out of focus of the objective lens (dashed line in Figure 10.2a), but to allow optically sectioned images to be acquired (Conchello and Lichtman 2005). Conversely, the development of mode-locked titanium:sapphire (Ti:Sa) laser techniques (Spence et al. 1991) largely contributed to the establishment of a two-photon excitation LSM (TPLSM) technique.