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
]. An MPM uses nonlinear optical interaction of biological molecules with a near-infrared, short pulse laser to provide deep tissue, 3D sectioning capability. Although 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 more effective strategy to increase the imaging depth is by using a longer excitation wavelength [3
Successful clinical application of optical imaging systems, such as optical coherent tomography, is attributed partially to their fiber-based implementation which makes the system compact and portable. For the clinical application of MPM systems, the compact and portable feature is essential. Ti: Sapphire-based MPM systems are usually bulky and difficult to transport. Several new kinds of fs laser sources are emerging, such as the passive mode-locking lasers using saturated Bragg reflectors, the optically pumped semiconductor laser, and optical parametric oscillators [6
]. There have been a number of drawbacks that prevent these lasers from becoming widely used MPM laser sources [6
]. The fiber-based femtosecond-pulsed (FBFP) laser is a promising source for MPM applications because the FBFP laser is compact and robust. Furthermore, an FBFP laser could provide wavelength tunability based on soliton frequency shift in special fibers, such as a highly nonlinear fiber and a higher order mode fiber [7
]. Finally, an FBFP laser is more suitable for compact and endoscopic applications because the fiber output feature allows direct coupling with fiber-based systems.
Efficient generation of a nonlinear signal requires focus of the excitation laser in both space and time. Fiber-based femtosecond-pulse (FBFP) sources that can deliver short fs pulses (<100 fs) at a high repetition rate (100 MHz) with relatively large average power (>500 mW) are essential for moving this technology from bench top to bed side. Although MPM systems based on commercially available FBFP lasers have been demonstrated by several groups [9
], none of them have demonstrated a fiber-based probe combined with a fiber laser source.
MPM imaging will limit average power on biological tissue to less than 50 mW due to safety considerations and there are a few commercial sub 100 fs fiber lasers with an average power that approaches 50 mW. However, a fiber-based endoscopic MPM or an MPM with a handheld probe requires a fiber fs laser with a much higher average power because prechirping for dispersion control, coupling to the fiber, and probe optics all contribute to power loss. Assuming the following efficiency values (pre-chirping unit, 60%; coupling to fiber, 40%; and probe optics, 70%), there is an overall efficiency of 17%. Therefore, 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. Recently, Dr. Wise group reported a FBFP laser with pulse width of 80 fs and an average power of more than 2 W [12
In this paper, we report the development of a compact multiphoton microscopy system which integrates an FBFP laser, a double clad photonic crystal fiber (DCPCF) and a miniature handheld probe. The laser occupies a space of 60 × 45 × 24 cm, and it can be condensed into a much smaller space. The laser produces a 125 fs pulse width and more than 1 W average power. The DCPCF guarantees effective delivery of fs excitation pulse and efficient collection of multiphoton signals via its large diameter core and a high numerical aperture (NA) clad. The hand-held probe has a diameter of 16 mm.