Raman Microscopy and Imaging

Abstract

AbstractRaman microscopy and imaging couples the spatial resolution of optical microscopy with the spectral resolution and information content of Raman spectroscopy. Together, a powerful tool for the investigation of chemical, or molecular, heterogeneity is created. Raman microspectroscopy enables the nondestructive analysis of small quantities of sample and the identification of contaminants and inclusions in situ within a matrix – both of great importance and utility in forensic investigations. Perhaps more importantly, it has been recognized that the microheterogeneity of products has an influence on both product performance and appearance. In the analysis of complex, multiphase systems, optical microscopy contrast methods (the majority of which depend upon refractive index changes) are commonly employed to provide information on the structural organization of materials. However, information on the chemical organization of materials is then often based on conjecture. Raman microscopy is arguably the most easily applied of a family of techniques that use the innate spectral signatures of the components as a contrast element and hence provides a method for analyzing chemical heterogeneity in “real” systems.Typically, the information that would be appropriate from an investigation of chemical heterogeneity would be:the number of chemical components present;the pure spectrum of each component;spatially resolved concentration information for each individual component.Raman microscopy and imaging provides a tool to determine these important factors in complex, multiphase systems. In its simplest form, the Raman microspectroscopy experiment requires a laser to be focused down to a diffraction‐limited spot on a sample and the inelastically scattered light is collected from that point and analyzed. This generates a Raman spectrum, which is indicative of the identity and quantity of the molecular species present in that sample volume. The limiting spatial resolution that can be obtained is of the order of 1 µm in the lateral plane and 2 µm in the axial plane, the microscopes used lending themselves to a confocal optical arrangement that provides this level of depth discrimination.The information content is constrained to that available in the Raman vibrational spectra and so commonly molecules with distinct functional groups are clearly distinguished, whilst it is more difficult to distinguish specifically between molecules with very similar molecular structure and chemical functionality. It is also a commonly held belief that Ramaninvestigations are hampered by fluorescence interference, which obscures the Raman spectrum. However, with modern Raman microscopes there are few samples where this limitation becomes completely defeating.Several approaches to the generation of Raman images (images where the contrast is based on the chemical or molecular heterogeneity) have been established and are discussed in detail. A comparison is made between these different Raman imaging approaches and also with different spectral imaging methods.