Optimizing Fluorescence Imaging Performance of Wide-Field Temporal-Focusing Microscopy for Multifluorophore-labeled Bio-specimens

Yu-Min Cheng^1*, Chi-Hsiang Lien², Chun-Yu Lin³, Chia-Yuan Chang⁴, Fan-Ching Chien¹

¹Department of Optics and Photonics, National Central University, Taoyuan, Taiwan
²Department of Mechanical Engineering, National United University, Miaoli, Taiwan
³College of Photonics, National Chiao Tung University, Tainan, Taiwan
⁴Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan, Taiwan

* Presenter:Yu-Min Cheng, email:u70958@gmail.com

The optical configuration of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have the optimal imaging performance. The optimal TPE absorption of fluorophores can be obtained for TPE images using a TFMPEM system. This study also aims to quantitatively evaluate the diffraction efficiency, excitation area, illumination power, and axial resolution of a TFMPEM system using different diffraction gratings, collimating lenses, excitation wavelengths, and objectives to provide the optimal performance of TFMPEM imaging. As a result, the optimal excitation area and axial sectioning of TFMPEM imaging for measuring the multifluorophore-labeled bio-specimens are achieved. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated.

Keywords: temporal-focusing multiphoton excitation microscopy, two-photon excitation, cell, mouse muscle fibers