Search

Tissue physiology and pathology can be better understood using two-photon microscopy that allows tissue structure and biochemistry to be assayed on the subcellular level. Histological analysis provides high-resolution tissue characterization and is the only imaging method that can be applied to tissues of arbitrary thicknesses. Scanning confocal microscopy was invented in 1962 by Minsky demonstrating that three-dimensional (3-D) resolved images can be obtained in translucent specimens without physical sectioning. An alternative 3-D imaging technology is two-photon excitation microscopy, which was introduced by Denk, Webb, and coworkers in 1990. The electronic transition of a fluorophore can be induced by the simultaneous absorption of two photons. Depth discrimination is the most important feature of two-photon microscopy. The design of a typical two-photon fluorescence microscope design is based on three basic components: a femtosecond laser system, high-throughput microscope optics, and high-sensitivity detection optoelectronics. An important advantage of two-photon imaging is its suitability for in vivo imaging.