High-resolution imaging in two-photon excitation microscopy using in situ estimations of the point spread function

Atsushi Doi, Ryosuke Oketani, Yasunori Nawa, Katsumasa Fujita

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

We present a technique for improving the spatial resolution of two-photon excitation microscopy, our technique combines annular illumination with an in situ estimation of the point spread function (PSF) used for deconvolution. For the in situ estimation of the PSF, we developed a technique called autocorrelation scanning, in which a sample is imaged by the scanning of two excitation foci that are overlapped over various distances. The image series obtained with the variation of the distance between the two foci provides the autocorrelation function of the PSF, which can be used to estimate the PSF at specific positions within a sample. We proved the principle and the effectiveness of this technique through observations of a fluorescent biological sample, and we confirmed that the improvement in the spatial resolution was ~1.7 times that of typical two-photon excitation microscopy by observing a mouse brain phantom at a depth of 200 μm.

Original languageEnglish
Article number#305380
Pages (from-to)202-213
Number of pages12
JournalBiomedical Optics Express
Volume9
Issue number1
DOIs
Publication statusPublished - Jan 1 2018
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Atomic and Molecular Physics, and Optics

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