title: Optimizing fluorescence excitation and detection for intravital two-photon microscopy.
abstract: Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra. Furthermore, the spectral characteristics of fluorescent proteins in vivo can be affected by the local environment and light scattering by deep tissue and can vary greatly from one laboratory to the next. Here, we describe a simple protocol for determining the two-photon excitation peaks of fluorescent reporters that can be tailored to the relevant tissue samples to suit the imaging goals of individual biological laboratories.
authors: Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
authors: Dan
authors: D
authors: Suan
authors: Henry R
authors: HR
authors: Hampton
authors: Michio
authors: M
authors: Tomura
authors: Osami
authors: O
authors: Kanagawa
authors: Tatyana
authors: T
authors: Chtanova
authors: Tri Giang
authors: TG
authors: Phan
mesh_terms: D000818:Animals; D002138:Calibration; D058948:Cell Tracking / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D008198:Lymph Nodes / Q000166:cytology; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D059629:Signal-To-Noise Ratio; D012984:Software
chemical_list: D049452:Green Fluorescent Proteins