Label-free multi-photon imaging using a compact femtosecond fiber laser mode-locked by carbon nanotube saturable absorber

K Kieu,N Peyghambarian,R A Norwood,R Gowda,S Mehravar

doi:10.1364/boe.4.002187

Abstract

We demonstrate label-free multi-photon imaging of biological samples using a compact Er(3+)-doped femtosecond fiber laser mode-locked by a single-walled carbon nanotube (CNT). These compact and low cost lasers have been developed by various groups but they have not been exploited for multiphoton microscopy. Here, it is shown that various multiphoton imaging modalities (e.g. second harmonic generation (SHG), third harmonic generation (THG), two-photon excitation fluorescence (TPEF), and three-photon excitation fluorescence (3PEF)) can be effectively performed on various biological samples using a compact handheld CNT mode-locked femtosecond fiber laser operating in the telecommunication window near 1560nm. We also show for the first time that chlorophyll fluorescence in plant leaves and diatoms can be observed using 1560nm laser excitation via three-photon absorption.

Highlights

Multi-photon (MP) imaging is a powerful technique that allows three dimensional mapping of samples that have a measurable nonlinear optical response such as SHG, THG or fluorescence induced by MP absorption
The benefits of using ultrafast Er3+-doped sources are many including the following: 1) the cost of the carbon nanotube modelocked excitation laser will be much lower; 2) longer wavelength operation may lead to larger imaging depth; 3) excitation with longer wavelengths reduces photo-toxicity of the sample; 4) THG imaging [27,28,29], an important label-free technique, can be implemented quite since the emission wavelength is in the visible where standard collection optics and photomultiplier tubes (PMT) for detection work best; 5) the laser beam can be delivered to the microscope using optical fiber, reducing the alignment effort; and, 6) this is an excitation wavelength that has not been thoroughly explored for MPI, providing the opportunity for new discovery
Work [6] on the THG imaging of plant leaves with a 1560nm excitation wavelength did not reveal this signal, so we believe that this is the first time that 3-photon excitation fluorescence of chlorophylls has been reported at this excitation wavelength

Summary

Introduction

Multi-photon (MP) imaging is a powerful technique that allows three dimensional mapping of samples that have a measurable nonlinear optical response such as SHG, THG or fluorescence induced by MP absorption. The benefits of using ultrafast Er3+-doped sources are many including the following: 1) the cost of the carbon nanotube modelocked excitation laser will be much lower (an order of magnitude below that of Ti:sa lasers); 2) longer wavelength operation may lead to larger imaging depth; 3) excitation with longer wavelengths reduces photo-toxicity of the sample; 4) THG imaging [27,28,29], an important label-free technique, can be implemented quite since the emission wavelength is in the visible where standard collection optics and photomultiplier tubes (PMT) for detection work best; 5) the laser beam can be delivered to the microscope using optical fiber, reducing the alignment effort; and, 6) this is an excitation wavelength that has not been thoroughly explored for MPI, providing the opportunity for new discovery

Multiphoton microscope and excitation laser

Results and discussion

Conclusions and outlook