Improved laser scanning fluorescence microscopy by multiphoton excitation

Publisher Summary The compound optical microscope has been central to countless advances in biological research. The growth of cell and molecular biology in recent years has forced the pace of microscopy research to provide high contrast images of increasingly more specific structures and functions in biological specimens. One technique is fluorescence microscopy, where highly specific chemical probes are used to stain particular components of the sample and imaged by their characteristic emission spectra. The method has advanced rapidly through parallel developments in optical filters and lenses, laser and nonlaser light sources, and highly efficient detectors. Fluorescence microscopy is able to deliver the high contrast needed to distinguish the fluorescence light of stained structures from a background of unwanted nonspecific fluorescence and reflected light features by the use of high-specification interference filters. Normally, a fluorescent molecule will absorb a single photon of the illuminating light and be excited to a highly energetic state. After a short but random period of time, some of this energy is lost as the molecule relaxes to a slightly lower energy before the molecule returns to the original or ground state by emitting a photon of light.