Multiphoton microscopy system with a compact fiber‐based femtosecond‐pulse laser and handheld probe

Gangjun Liu,Khanh Kieu,Zhongping Chen,Frank W Wise

doi:10.1002/jbio.201000049

Abstract

We report on the development of a compact multiphoton microscopy (MPM) system that integrates a compact and robust fiber laser with a miniature probe. The all normal dispersion fiber femtosecond laser has a central wavelength of 1.06 μm, pulse width of 125 fs and average power of more than 1 W. A double cladding photonic crystal fiber was used to deliver the excitation beam and to collect the two-photon signal. The hand-held probe included galvanometer-based mirror scanners, relay lenses and a focusing lens. The packaged probe had a diameter of 16 mm. Second harmonic generation (SHG) images and two-photon excited fluorescence (TPEF) images of biological tissues were demonstrated using the system.

Highlights

Multiphoton microscopes (MPM), which include two-photon excited fluorescence (TPEF) microscopy and second harmonic generation (SHG) microscopy, have been widely used for biological imaging with high contrast and submicron resolution [1, 2]
An MPM provides a larger penetration depth compared to a confocal microscope, the penetration depth is limited to around 600 μm for typical multiphoton systems which use the Ti: Sapphire femtosecond laser with an excitation wavelength of around 800 nm and repetition rate of around 80 MHz
We report the development of a compact multiphoton microscopy system which integrates an fiber-based femtosecond-pulsed (FBFP) laser, a double clad photonic crystal fiber (DCPCF) and a miniature handheld probe

Summary

Introduction

Multiphoton microscopes (MPM), which include two-photon excited fluorescence (TPEF) microscopy and second harmonic generation (SHG) microscopy, have been widely used for biological imaging with high contrast and submicron resolution [1, 2]. An MPM provides a larger penetration depth compared to a confocal microscope, the penetration depth is limited to around 600 μm for typical multiphoton systems which use the Ti: Sapphire femtosecond (fs) laser with an excitation wavelength of around 800 nm and repetition rate of around 80 MHz. Imaging depth of MPM may be increased by increasing the average power, decreasing the pulse width or decreasing the repetition rate of the laser. A fiber fs laser with pulse width of sub 100 fs, and an average power of larger than 300 mW will be an ideal source for endoscopic MPM applications. Such a source is currently not commercially available. The hand-held probe has a diameter of 16 mm

System setup

Fiber laser

Handheld probe design

Data acquisition and software

Results

Conclusions