Nonlinear Optical Contrast Research Articles

High volume confinement in two-photon total-internal-reflection fluorescence correlation spectroscopy

We report results on two-photon total-internal-reflection fluorescence correlation spectroscopy with radially polarized light. The combination of liquid crystal spatial light modulator, providing radial polarization with ultrafast laser (picosecond Nd:GdVO4 laser), allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

Nonlinear optical microscopy of the bronchus

Because of frequent exposure to carcinogens, the bronchus is prone to early pathologic alterations. The assessment of these early changes is of key significance in physiological studies and disease diagnosis of the bronchus. We utilize nonlinear optical microscopy (NLOM) to image mouse bronchial tissue based on intrinsic nonlinear optical contrast. Our results show that NLOM is effective for imaging the bronchial intact microstructural components, providing quantitative information about the biomorphology and biochemistry of tissue. Our findings also display that NLOM can provide a two-photon ratiometric redox fluorometry, based on mitochondrial signals and reduced pyridine nucleotide (NADH and NADPH) and oxidized flavoproteins (Fp) signals, to assess the metabolic state of the epithelial cells and chondrocytes. It was found that NLOM can offer a sensitive tool, based on the second-harmonic signal depth-dependent decay, to obtain quantitative information on the optical property of the stroma associated with normal and diseased tissue states. Our results suggest that with the advent of the clinical portability of typical nonlinear optical endoscopy, the NLOM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of bronchial disease.

Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy

Arterial tissues collected from Ossabaw swine bearing metabolic syndrome-induced cardiovascular plaques are characterized by multimodal nonlinear optical microscopy that allows coherent anti-Stokes Raman scattering, second-harmonic generation, and two-photon excitation fluorescence imaging on the same platform. Significant components of arterial walls and atherosclerotic lesions, including endothelial cells, extracellular lipid droplets, lipid-rich cells, low-density lipoprotein aggregates, collagen, and elastin are imaged without any labeling. Emission spectra of these components are obtained by nonlinear optical microspectrometry. The nonlinear optical contrast is compared with histology of the same sample. Multimodal nonlinear optical imaging of plaque composition also allows identification of atherosclerotic regions that are vulnerable to rupture risk. The demonstrated capability of nonlinear optical microscopy for label-free molecular imaging of atherosclerotic lesions with 3-D submicrometric resolution suggests its potential application to the diagnosis of atherosclerotic plaques, determination of their rupture risk, and design of individualized drug therapy based on plaque composition.