title: Living cell functions and morphology revealed by two-photon microscopy in intact neural and secretory organs.
abstract: Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon microscopy, has enabled the quantification of spatiotemporal patterns of [Ca(2+)](i) and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocytosis" for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.
authors.affiliation: Supportive Center for Brain Research, National Institute for Physiological Science and the Graduate University for Advanced Studies (SOKENDAI), 38 Nishigonaka, Okazaki, Aichi 444-8585, Japan. tn@nips.ac.jp
authors.forename: Tomomi
authors.initials: T
authors.lastname: Nemoto
mesh_terms: D000818:Animals; D005089:Exocytosis / Q000502:physiology*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D046790:Pancreas, Exocrine / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25 / Q000502:physiology
chemical_list: D050825:Synaptosomal-Associated Protein 25