Saturated excitation microscopy using differential excitation for efficient detection of nonlinear fluorescence signals

Yasunori Nawa, Yasuo Yonemaru, Atsushi Kasai, Ryosuke Oketani, Hitoshi Hashimoto, Nicholas I. Smith, Katsumasa Fujita

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

We report a method to increase the efficiency of detecting nonlinear fluorescence signals in saturated excitation (SAX) microscopy. With this method, we compare fluorescence signals obtained under different degrees of saturated excitation to extract the nonlinear fluorescent signal induced by saturated excitation. Compared to conventional SAX microscopy using the harmonic demodulation technique, the detection efficiency of the fluorescence signal can be increased up to 8 and 32 times in imaging using the second-order and the third-order nonlinear fluorescence signals, respectively. We combined this approach with pulsed excitation, which is effective to reduce photobleaching effects, and achieved super-resolution imaging using third-order nonlinear fluorescence signals induced by saturated excitation of an organic dye. The resolution improvement was confirmed in the observations of fluorescent beads, actin-filaments in HeLa cells, and a spine in mouse brain tissue.

Original languageEnglish
Article number080805
JournalAPL Photonics
Volume3
Issue number8
DOIs
Publication statusPublished - Aug 1 2018
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Atomic and Molecular Physics, and Optics
  • Computer Networks and Communications

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