Abstract
New system for a wide-field CARS microscopy is demonstrated, including two schemes of non-phase-matching illumination. Several advantages including high Stokes pulse energy, pulse-to-pulse stability and inherent synchronization between pump and Stokes pulses were brought by use of methane-filled Raman converter. Spatial resolution of the system with axially symmetric illumination, 0.5 microm, was found to correspond to diffraction limit of the imaging objective. Selective sensitivity to lipid-rich myelin sheaths in the nerve tissue has been demonstrated and confirmed by comparison with histological samples stained with myelin-specific dye. Single-shot imaging capability of the system has been demonstrated with a speckling-free illumination on a monolayer of 3 microm polystyrene beads.
Highlights
Contrast mechanisms currently utilized in optical microscopy can be grouped into two general categories: exogenous staining associated with specific markers, and intrinsic optical properties of the tissue
Several advantages including high Stokes pulse energy, pulse-to-pulse stability and inherent synchronization between pump and Stokes pulses were brought by use of methane-filled Raman converter
Selective sensitivity to lipid-rich myelin sheaths in the nerve tissue has been demonstrated and confirmed by comparison with histological samples stained with myelin-specific dye
Summary
Contrast mechanisms currently utilized in optical microscopy can be grouped into two general categories: exogenous staining associated with specific markers, and intrinsic optical properties of the tissue. Both approaches have a wide range of applications, and continue to progress in many fields of biological and medical research. Long wavelengths of mid-IR radiation limit the spatial resolution to several micrometers, and are difficult to use with aqueous samples due to strong water absorption These issues are circumvented in Raman microscopy, which utilizes visible and near-IR radiation to detect spectral shifts related to specific molecular vibrations. It is often difficult to separate Stokes radiation from the fluorescent background since both types of emission are shifted towards longer wavelengths
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