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

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

doi:10.1364/boe.9.003449

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.

Highlights

As optical imaging becomes crucial in the medical and life sciences, there has been a leap forward in biomedical imaging techniques over the past decade
The lifetime signal exhibited around F-actin labelled with Alexa Fluor 488, mitochondria labelled with Red CMXRos, and nucleus labelled with DAPI were shown to be slightly different from each other (~0.9 ns, ~0.7 ns, and ~1.1 ns, respectively) as different type of fluorophores will have different values of lifetime
Even in snap shot image with short pixel dwell time (85 ns), the measured fluorescence lifetime is shown to be almost identical as the one with longer dwell times (255 ns, 425 ns, and 2500 ns at 3, 5, and 30 averaging, respectively), shows that the performance of our fluorescence lifetime imaging microscopy (FLIM) system is high enough to capture the lifetime at low photon rate

Summary

Introduction

As optical imaging becomes crucial in the medical and life sciences, there has been a leap forward in biomedical imaging techniques over the past decade. Nicotinamide adenine dinucleotide (NADH)) based on the principle that fluorescence lifetime varies depending on the local chemical density or composition [2,3,4,5,6,7,8]. Biochemical effectors such as signaling molecules or proteins are known to have their primary function as computational elements and to build various compartmentalized biochemical circuits within living cells and tissues [9]. The major obstacle utilizing FLIM in real-time imaging was the extensively long data acquisition and processing time

Methods

Results

Conclusion