Research Papers: Imaging

Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

[+] Author Affiliations
Chi-Hsiang Lien

National United University, Department of Mechanical Engineering, Miaoli, Taiwan

Gerald Abrigo, Pei-Hsuan Chen, Fan-Ching Chien

National Central University, Department of Optics and Photonics, Taoyuan, Taiwan

J. Biomed. Opt. 22(2), 026008 (Feb 23, 2017). doi:10.1117/1.JBO.22.2.026008
History: Received November 20, 2016; Accepted February 8, 2017
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Abstract.  Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore–labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830  lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7  μm and the shifting distance of the temporal focal plane was less than 0.95  μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

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© 2017 Society of Photo-Optical Instrumentation Engineers

Citation

Chi-Hsiang Lien ; Gerald Abrigo ; Pei-Hsuan Chen and Fan-Ching Chien
"Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation", J. Biomed. Opt. 22(2), 026008 (Feb 23, 2017). ; http://dx.doi.org/10.1117/1.JBO.22.2.026008


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