Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging

Ryosuke Oketani, Haruka Suda, Kumiko Uegaki, Toshiki Kubo, Tomoki Matsuda, Masahito Yamanaka, Yoshiyuki Arai, Nicholas Smith, Takeharu Nagai, Katsumasa Fujita

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)1-5
Number of pages5
JournalJournal of Biomedical Optics
Issue number1
Publication statusPublished - Nov 1 2019
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Biomedical Engineering


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