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Adapting a compact confocal microscope system to a two‐photon excitation fluorescence imaging architecture
Author(s) -
Diaspro Alberto,
Corosu Mirko,
Ramoino Paola,
Robello Mauro
Publication year - 1999
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/(sici)1097-0029(19991101)47:3<196::aid-jemt5>3.0.co;2-v
Subject(s) - microscope , optics , confocal , fluorescence , laser , materials science , physics
Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two‐photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode‐locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high‐power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single‐pinhole design. Three‐dimensional point‐spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura‐2, Indo‐1, DiOC 6 (3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two‐photon mode. Microsc. Res. Tech. 47:196–205, 1999. © 1999 Wiley‐Liss, Inc.