Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.
Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.
['Oketani|Ryosuke|R|', 'Suda|Haruka|H|', 'Uegaki|Kumiko|K|', 'Kubo|Toshiki|T|', 'Matsuda|Tomoki|T|', 'Yamanaka|Masahito|M|', 'Arai|Yoshiyuki|Y|', 'Smith|Nicholas|N|', 'Nagai|Takeharu|T|', 'Fujita|Katsumasa|K|']
D005456:Fluorescent Dyes D006056:Golgi Apparatus D006367:HeLa Cells D006801:Humans D021621:Imaging, Three-Dimensional D036641:Microscopy, Fluorescence, Multiphoton D009068:Movement D055620:Optical Phenomena D059010:Single-Cell Analysis D059008:Time-Lapse Imaging
two-photon excitation multifocus excitation spinning disk confocal microscopy super-resolution microscopy three-dimensional imaging