Developing Three-Photon-Induced Fluorescence Microscopy using Tryptophan

Abstract

Multi-photon fluorescence microscopy is a useful tool for whole cell and in-vivo tissue imaging. That is because the near-infrared (NIR) excitation laser pulses provides deep tissue imaging capbility, a low backgournd signal due to a large spectral separation from the wavelength of the fluorescence, and 3D image reconstruction capability. It is particulary advantageous when fluoresce wavelength of a fluorophore is so short that the UV excitation is required. In this presentation, we report the recent progress in three-photon-induced fluorescence (3PIF) microscopy on tryptophan (Trp), one of the aminoacids. The Trp fluorescences at 350 nm upon the 280-nm excitation. It has been a strong cadicate as a intrinsic fluorophore for biological fluorescence imaging. The 3PIF scheme allows non-UV excitation, which increases viablity of cells and tissues during the measurement and low non-sepcific autofluorescence from the biological systems. A 3PIF microscopy was home-built to maximize the transimission of the NIR excitation laser pulses and collection efficiency of the UV fluorescence. The fluorescence intensity vs. excitation power curve was used to monitor the three-photon absorption process and optimize the optical setup. The point spread function measurement using fluorescent beads (100-nm dia., λabs= 260 nm, λem=450 nm) showed that our home-built 3PIF microscope has 1.21-µm lateral resolution and 6.73-µm axial resolution at 980-nm excitation through 60X objective lens (water immersion, NA 1.2). We also present a 3D image of glass beads in Trp monomer solution to demonstrate 3D-imaging capability of the 3PIF microscopy.