Laser sources have been essential for the advances in CARS microscopy. In the 1980s, visible dye lasers with non-collinear beam geometry were used in the first CARS microscope [21
]. In 1999, Xie and coworkers revived this technique by using two synchronized near infrared fs pulse trains for CARS imaging in a collinear beam geometry [22
]. Later, Hashimoto et al.
used an amplified laser system to produce two ps pulse trains for CARS imaging [9
]. Cheng et al.
indicated for the first time that tunable ps lasers operating in the NIR wavelengths provides not only high spectral resolution, but also superior vibrational contrast over fs lasers [7
]. In the spectral domain, the spectral width of a fs pulse is much broader than the width of most Raman lines, i.e. vibrational line widths are typically on the order of 10 cm−1
whereas fs pulses are around 100 cm−1
in bandwidth. On the other hand, the spectral width of a ps pulse matches the Raman line width, thus focusing the excitation energy on a single Raman band and permitting high-speed CARS imaging. Since 2001, ps laser sources have been widely used in developments of CARS microscopy, including electronically synchronized Ti:sapphire lasers [23
] and synchronously pumped, intracavity-doubled ps OPO [24
]. In parallel, various designs based on fs lasers were proposed to utilize the advantages of fs pulses. CARS microscopy with a single broadband source through optical pulse shaping was demonstrated [25
]. Multiplex CARS microscopy with ps/fs pulse excitation [27
] and with a laser-pumped photonic crystal fiber have been extensively explored [30
]. However, high-speed and high-quality images were still difficult to obtain with these methods. Moreover, it is difficult to perform multimodality imaging on these platforms.
The current work couples CARS microscopy to a widely used multiphoton imaging platform based on a fs laser, a synchronously pumped fs OPO, and a PPLN doubling crystal. This method provides a cost-efficient way to maximize the bioimaging capabilities of NLO microscopy. It also offers several advantages over multimodal imaging with two synchronized ps lasers [14
]. First, all the pulses are inherently synchronized, which eliminates the need for day-to-day alignment of temporal overlapping of the two beams for CARS imaging. Second, all the wavelengths are in the near IR region from 700 nm to 1.3 μm, a golden window for tissue imaging. Third, the fs pulses allow efficient generation of TPF, SHG, and THG signals. Importantly, unlike amplified fs lasers [22
], the pulses of a high repetition rate in our system permit high-speed imaging. Although the highest acquisition rate of our microscope is 2 μs/pixel, video rate imaging using advanced scanning configurations [24
] is feasible due to the significant NLO signal level. Furthermore, the three-beam modality with tunable ability allows background free CARS imaging via time-resolved detection.
Compared with ps lasers, a disadvantage associated with fs lasers is the higher peak power at the focus. It was shown that the photodamage in CARS microscopy [35
] increases with shorter wavelengths. In our system, the average power of the 1018 nm beam at the sample is relatively low (2.2 mW), and thus a higher power (10 to 20 mW) of 790 nm beam is used. Increasing the power of the Stokes beam by intracavity doubling would be beneficial to increase the photodamage threshold and further enhance the vibrational contrast by minimizing the two-photon resonance enhancement of the non-resonant background.
To summarize, we have demonstrated multimodal NLO imaging based on a turn-key fs laser, an OPO, and a frequency doubling system. Our method provides a cost-effective solution to implement CARS and THG imaging on a widely used multiphoton microscope. The integration of CARS, SHG, THG, and multiphoton fluorescence on the same microscope platform greatly enhances the capability, applicability, and versatility of NLO microscopy.