23 February 2017 Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation
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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    μ m and the shifting distance of the temporal focal plane was less than 0.95    μ m 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.
© 2017 Society of Photo-Optical Instrumentation Engineers (SPIE)
Chi-Hsiang Lien, Gerald Abrigo, Pei-Hsuan Chen, Fan-Ching Chien, "Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation," Journal of Biomedical Optics 22(2), 026008 (23 February 2017). https://doi.org/10.1117/1.JBO.22.2.026008 . Submission: Received: 20 November 2016; Accepted: 8 February 2017
Received: 20 November 2016; Accepted: 8 February 2017; Published: 23 February 2017
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