An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

Yu Min Cheng, Chi Hsiang Lien, Jing Han Ke, Fan Ching Chien

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3-5.0 μm for excitation wavelengths of 730-1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.

Original languageEnglish
Article number235401
JournalJournal of Physics D: Applied Physics
Volume53
Issue number23
DOIs
StatePublished - 3 Jun 2020

Keywords

  • dual-color
  • excitation wavelength switching
  • temporal-focusing multiphoton excitation microscopy
  • two-photon excitation spectrum

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