Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser

Jiheun Ryu; Ungyo Kang; Jayul Kim; Hyunjun Kim; Jue Hyung Kang; Hyunjin Kim; Dae Kyung Sohn; Jae-Heon Jeong; Hongki Yoo; Bomi Gweon

doi:10.1364/BOE.9.003449

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser

Biomed Opt Express. 2018 Jun 28;9(7):3449-3463. doi: 10.1364/BOE.9.003449. eCollection 2018 Jul 1.

Authors

Jiheun Ryu^{1

2}, Ungyo Kang³, Jayul Kim¹, Hyunjun Kim¹, Jue Hyung Kang³, Hyunjin Kim⁴, Dae Kyung Sohn⁵, Jae-Heon Jeong¹, Hongki Yoo^{3

6}, Bomi Gweon^{3

7}

Affiliations

¹ Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
² Wellman Center for Photomedicine, Harvard Medical School & Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
³ Department of Biomedical Engineering, Hanyang University, Seoul 04763, South Korea.
⁴ Molecular Imaging & Therapy Branch, Research Institute and Hospital, National Cancer Center, Goyang, 10408, South Korea.
⁵ Innovative Medical Engineering & Technology, Division of Convergence Technology, Research Institute and Hospital, National Cancer Center, Goyang, 10408, South Korea.
⁶ hyoo@hanyang.ac.kr.
⁷ gweonbomi@gmail.com.

Abstract

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

Keywords: (110.6880) Three-dimensional image acquisition; (170.3650) Lifetime-based sensing; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy.