Resolution-Enhanced Coherent Nonlinear Microscopy of Plant Tissues

Biological imaging with molecular specificity is most often achieved using fluorescent labeling. Fluorescent light for image contrast is also the basis of most optical “super-resolution” techniques that achieve far-field spatial resolution beyond the classical limit. However, non-invasive imaging techniques have been developed that use only native fluorophores, or bypass fluorescence in favor of other forms of inherent (endogenous) contrast. Molecular identification can be done endogenously by probingthe Raman spectrum, although this is a weak process and requires coherent methods to enhance the signal using pulsed lasers. To date, non-fluorescent super-resolution imaging methods that target coherent nonlinear processes such as coherent Raman spectroscopy and harmonic generation have been exceedingly rare and demonstrated limited effectiveness. We will build an imaging system for examining endogenous contrast mechanisms in biological samples, with chemical specificity, achieving super-resolution for all contrast mechanisms including coherent Raman scattering. We will employ a novel use of an imaging scheme known as multiphoton spatial- frequency modulated imaging. In Phase I, we will build the microscope and characterize its imaging performance. Performance will be measured with test targets and compared to numerical models. We will demonstrate resolution-enhanced biological imaging of endogenous fluorophores, coherent Raman signals, and harmonic generation signals. We plan to target structures that are relevant to metabolic processes with applications toward biofuel generation. Our proposed instrument will provide a valuable tool enabling biological research to proceed with enhanced spatial resolution for a variety of contrast mechanisms beyond fluorescence. Our technology can be added as a bolt-on module to the front end of existing multiphoton microscopes, or be built as a stand-alone instrument. The technology is relatively insensitive to wavelength, and can be used toprovide enhanced resolution of nonlinear signals from the UV to the mid-infrared. Other application spaces for our instrument include materials science, advanced manufacturing, biomedical research, and homeland security.