Broadly Tunable High-Energy Spectrally Focused CARS Microscopy with Chemical Specificity and High Resolution for Biological Samples

摘要

The current trend in laser sources for Coherent Anti-Stokes Raman Scattering (CARS) microscopy consists of picosecond optical parametric oscillators (OPO)s and femtosecond-pumped fiber supercontinuum sources. While both methods are proven CARS performers, restricted wavelength tuning range and low power limit the Raman lines and types of samples that may be practically interrogated. To address these limitations, we present a novel, highly tunable spectrally focused femtosecond Optical Parametric Amplifier (OPA) and microscope system optimized for CARS microscopy. The laser source consists of an amplified ytterbium fiber laser driving a pair of OPAs producing two outputs that produce tunable femtosecond pulses from 650 to 1300nm. Each OPA may be tuned independently of the other over its entire range, allowing the addressing of any arbitrary wavenumber from 0 to 7700 cm~(-1). Additionally, the complete freedom of tuning allows one beam to be set at the optimal wavelength for a complementary technique, such as two-photon fluorescence or second harmonic, while the second beam is then tuned to the desired wavenumber difference for CARS. The femtosecond pulses are chirped out to the picosecond regime, reducing non-resonant background and providing improved spectral resolution. Typically, OPA systems are limited to kHz repetition rates, making them impractical for imaging applications. In contrast, our OPA system is driven at 1 MHz, providing a sufficient pulse rate for high-resolution imaging at rates of 1-2 frames per second. The 1 MHz rate preserves good pulse energy while reducing average power, thus limiting sample photo damage.