This award is for developing improved contrast in Coherent Anti-Stokes Raman Scattering [CARS] Microscopy through Focus-Engineering [FE-CARS]. Coherent anti-Stokes Raman scattering (CARS) microscopy has rapidly ascended as a noninvasive tool for imaging cells and tissues with chemical selectivity. Based on its unique vibrational selectivity to lipids, proteins, and DNA, the full range of potential CARS applications to biological problems is vast. Nonetheless, due to the presence of a ubiquitous nonresonant background and the weak vibrational response of several molecular groups, applications have been limited to imaging of lipids so far. To open up a new range of applications for CARS microscopy in biology and biomedicine, background suppression and contrast improvement methods are imperative. This work introduces a new approach to CARS microscopy, which provides immediate contrast improvement. The approach is based on controlling the spatial interference of the CARS waves emanating from the focal volume. Emission control is achieved by spatial phase shaping, or focal engineering, of the incident laser pulses using a spatial light modulator (SLM). The use of appropriate phase masks allows controlled destructive interference of unwanted background contributions, while constructive interference accentuates vibrationally resonant objects. A focus-engineered (FE-) CARS microscope will be developed, with the following milestones: 1) Characterizing and optimizing contrast improvement with FE-CARS through computer-controlled phase shaping of the incident transverse beam profiles; 2) Combining FE-CARS with two-photon excited fluorescence (TPEF) and focus-engineered second harmonic generation (SE-SHG) for multimodal imaging of tissues and cells. FE-CARS multimodal imaging will be tested on a) lipid depositions and calcium hydroxyapapatite crystals; b) structural changes to collagen fibers under the influence of glycerol or dimethyl-sulfoxide.
The proposed microscope development is anticipated to have far reaching implications for biological imaging studies. The proposed instrument will be used for training graduate students, undergraduate students and pre-college students in the field of nonlinear microscopy. The instrument will be used in several outreach programs. Among these programs, the PI organized the Minority Science Program of the School of Biological Sciences at the university and a 4-week Summer School for high school students.
