Volumetric Raman Spectral Imaging With a Confocal Raman Microscope: Image Modalities and Applications

Abstract Confocal Raman microscopy is an invaluable tool for non-invasive three-dimensional (3-D) analysis and visualization of chemically heterogeneous samples. The confocal Raman microscope (CRM) combines focused laser illumination with spatially filtered detection to define in the sample a localized volume element (voxel) from which scattered or reflected light is detected. Attenuation of light from outside the voxel by the pinhole spatial filter increases image contrast and results in voxel sizes typically <10−18 m3. Moving the specimen relative to the voxel and recording the scattered light Raman spectrum at each scan position generates a Raman spectrally-encoded 3-D sample image for non-invasive in situ differentiation between optically similar yet chemically distinct materials. The motivation for the present work stems from our application of continuum modeling techniques as the mathematical framework for development of physiologically realistic biosystem models. A Type 2 correlation-based confocal scanning laser microscope (CSLM) integrated with a high performance, parallel-drive micro-motion robot (MR-1) was built for spatially-distributed acquisition of optical and mechanical data within a < 1 mm3 workspace volume.