Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation)

Hiroki Segawa,Philippe Leproux,Masanari Okuno,Vincent Couderc,Hideaki Kano,Hiro-O Hamaguchi

doi:10.1364/oe.20.009551

Abstract

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

Highlights

Multimodal nonlinear optical imaging is widely used to become one of the most powerful methods to analyze biological systems such as living cells and tissues [1,2,3,4,5,6]
We have developed a new multimodal molecular imaging system that combines coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), third harmonic generation (THG) and multiplex third-order sum frequency generation (TSFG) using a subnanosecond white-light laser source
Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG

Summary

Introduction

Multimodal nonlinear optical imaging is widely used to become one of the most powerful methods to analyze biological systems such as living cells and tissues [1,2,3,4,5,6]. A Ti:Sapphire laser oscillator is often employed for multimodal nonlinear optical imaging in order to obtain high photon density at the focal position [2,3,4,6] With such a light source, we have to tune the laser wavelength to achieve resonances [6,9]. In the present experimental condition, the TSF signal is generated from the combination of a narrow-band laser (ω1) and a broadband white-light laser (ω2), with ωTSF = 2ω1 + ω2 and ω1 + 2ω2 Using this new multimodal imaging scheme, living cells are clearly visualized with different molecular contrasts through multiplex CARS, SHG, THG and multiplex TSFG simultaneously

Experimental

Results and discussion

Conclusion