Increasing brightness in multiphoton microscopy with a low-repetition-rate, wavelength-tunable femtosecond fiber laser

Abstract

Many experiments in biological and medical sciences currently use multiphoton microscopy as a core imaging technique. To date, solid-state lasers are most commonly used as excitation beam sources. However, the most demanding applications require precisely adjusted excitation laser parameters to enhance image quality. Still, the lag in developing easy-to-use laser sources with tunable output parameters makes it challenging. Here, we show that manipulating the temporal and spectral properties of the excitation beam can significantly improve the quality of images. We have developed a wavelength-tunable femtosecond fiber laser that operates within the 760–800 nm spectral range and produces ultrashort pulses (<70 fs) with a clean temporal profile and high pulse energy (~1 nJ). The repetition rate could be easily adjusted using an integrated pulse picker unit within the 1–25 MHz range and without strongly influencing other parameters of the generated pulses. We integrated the laser with a two-photon excited fluorescence (TPEF) scanning laser microscope and investigated the effect of the tunable wavelength and reducing the pulse repetition rate on the quality of obtained images. Using our laser, we substantially improved the images’ brightness and penetration depth of native fluorescence and stained samples compared with a standard fiber laser by manipulating the excitation beam’s temporal and spectral properties. Our results will contribute to developing imaging techniques using lower average laser power and broader use of tailored fiber-based sources.