Two-Photon Excitation Spectral and Imaging Measurement in Bio-tissue by Temporal Focusing Multiphoton Excitation Microscopy

Ching-Lung Luo^1*, Jian-Zong Lai¹, Yu-Min Cheng¹, Chi-Hsiang Lien², Fan-Ching Chien¹

¹Department of Optics and Photonics, National Central University, Taoyuan 32001, Taiwan
²Department of Mechanical Engineering, National United University, Miaoli 36063, Taiwan

* Presenter:Ching-Lung Luo, email:a109226004@g.ncu.edu.tw

Fluorescence imaging currently is a major approach in the biomedical application field. Among the fluorescence microscopies, multiphoton excitation microscopy allows observing the distribution of the specific molecules labeled with different fluorophores in the thick biological tissue. Temporal focusing multiphoton excitation microscopy (TFMPEM) has the advantages of deeper penetration, optical section, minimized invasion, less photodamage, and better temporal resolution for in-vivo imaging. However, different labeling fluorophores have their own best two-photon excitation (TPE) efficiency in certain excitation wavelength. Thence, this study presented an excitation wavelength-switchable optical system in TFMPEM, allowed wavelength switching to obtain the best excitation efficiency for different labeling fluorophores. The presented system provided the switching of excitation wavelength in the range of 730 nm to 1000 nm and was examined by the vasculature of the mouse brain labeled with the fluorophore. Thus, the vasculature of the mouse brain was acquiring in different depths and visualize in the results of three-dimensional images. The TPE spectra of the different fluorophores also can be obtained through scanning different excitation wavelengths. Additionally, the nano-carrier uptake of the living cells via clathrin-mediated endocytosis was explored by two-color TFMPEM images.

Keywords: Multiphoton Excitation, Temporal Focusing, Fluorescence Microscopy, Bio-tissue