Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

Matthias Eibl,Sebastian Karpf,Tom Pfeiffer,Robert Huber,Daniel Weng,Jan Philip Kolb,Hubertus Hakert

doi:10.1364/boe.8.003132

Abstract

Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

Highlights

Nonlinear optical microscopy is a powerful technique in bio-molecular science that allows to gain a better understanding of biochemical processes on a cellular level
In contrast to time correlated single photon counting (TCSPC), the instrument response function (IRF) or apparatus function is given by the electronic bandwidth of the overall system in the SP-fluorescence lifetime imaging microscopy (FLIM) approach
We show that longer pulses in the many-10ps to nanosecond regime are suitable for two-photon excited fluorescence (TPEF) imaging and allow for fast FLIM imaging

Summary

Introduction

Nonlinear optical microscopy is a powerful technique in bio-molecular science that allows to gain a better understanding of biochemical processes on a cellular level. Different fluorophores may have different lifetimes, but more importantly, the lifetime of one and the same molecule can critically depend on its molecular environment. This can yield valuable information about electronic interactions, steric configuration and vibrational coupling [4]. With this wealth of information, TPEF microscopy combined with FLIM is a valuable tool for researchers throughout many fields of bio-molecular science [5,6,7]

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Conclusion