Development of High Contrast Coherent Anti-Stokes Raman Scattering (CARS) and Multiphoton Microscopy for Label-Free Biomolecular Imaging

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy has received much interest for imaging cells and tissues due to its outstanding capabilities of biochemical selectivity using molecular vibrations, high sensitivity, as well as intrinsic three-dimensional optical sectioning ability. In this thesis, the polarization effects in CARS microscopy have been comprehensively studied and thereby several novel CARS microscopic techniques for high contrast vibrational imaging and high sensitive molecular orientation detection have been reported. An advanced interferometric polarization CARS imaging technique was developed to effectively suppress the nonresonant background, while greatly enhance the weak resonant signals of low concentration biochemicals for high contrast and high sensitive biomolecular imaging. To further reduce the excitation power for minimizing the photodamage to the specimens, a unique heterodyne-detected polarization CARS technique by utilizing interference of the relatively intense local oscillator CARS signal and the weak resonant CARS signal generated simultaneously within the focal volume of the sample was also developed for high sensitive CARS imaging. In addition, employing an elliptically polarized pump field combined with a linearly polarized Stokes field, intrinsic background-free CARS imaging was realized with much higher resonant signal intensities to be detected as compared to conventional polarization CARS. To facilitate the three dimensional molecular orientation sensing, a radial polarization CARS microscope was demonstrated for improving the detection of longitudinally oriented molecules in the samples. Further, an integrated CARS and multiphoton microscopy technique by implementing a dual 4-f configured paired-gratings spectral filtering module on a dual-color femtosecond laser source has also been successfully developed for biomolecular imaging. It was demonstrated that high contrast CARS and high quality multiphoton microscopy imaging could be acquired in tandem on the same platform for quantitative assessment of biomolecular changes associated with liver disease transformations (e.g., fatty/fibrotic liver). This research indicated the great applicable potential of the integrated CARS microscopy and multiphoton microscopy for label-free biomolecular imaging in biological and biomedical systems.