A Raman microscope combines a Raman spectrograph and a light microscope to gain chemical and structural information from materials down to the micron scale. Raman spectroscopy observes when the wavelength of light changes as it interacts with a molecule. The different wavelengths seen in Raman scattering can identify and study vibrational, rotational, and bending forces within chemical bonds of a molecule.
Three dimensional (3-D) Raman imaging offers a look at materials unlike any other technique by creating a three-dimensional construction at the micron scale of the chemistry within the material. While other microscopies can examine the morphology of a sample, and other spectroscopies can identify the elemental constituents in the sample, Raman provides both structural and chemical information of microscopic samples. Infrared microspectroscopy cannot match the scale of Raman microscopy. Raman also yields molecular information from materials that have weak infrared signals such as double and triple bond carbon molecules. Additionally, Raman can examine samples in an aqueous mixture and sample through glass and clear plastic.
Optical sectioning in light microscopy produces images in a series of focal planes within a thick sample. Rather than physical sectioning of the sample, this enables the microscope viewer to non-destructively obtain thin slices from thick samples by removing out-of-focus light in each image plane. Confocal microscopy uses an illuminated sample spot and a pinhole aperture within the beam path share the same focal point. In practical terms, instead of the entire sample, only a small part is illuminated by a point-shaped light source. The pinhole then blocks unfocused light, thus increasing contrast and depth of field.
Frequently the chemistry of a solid material changes within the depth of that material, sometimes gradually and sometimes abruptly changing from one phase to another. Confocal Raman microscopy describes the ability of a Raman system to spatially filter the analysis volume of the sample, in the x, y (lateral) and z (depth) axes. This optical sectioning technique provides, non-destructively, Raman spectra from each focal plane it samples. A Raman imaging microscope enables the user to create a chemical map of each plane. 3D confocal visualization software then stitches each plane into a hyperspectral 3D representation of the sample. The data set can be recalled for analysis or spectral matching at any time post collection.
A significant challenge to confocal microscopy is handling spherical aberrations which occur when the focal point of light passing through the marginal regions of a lens that is different from the light passing through the central region of the lens. The spherical aberration effects amplify with increasing depth into the sample. When light is focused at points further into the sample than what is expected from the Z axis position of the stage alone, the resulting image will appear to be an artificially compressed. Also, a spreading of focal points creates a blurring in the focus, which results in both a loss of spatial resolution and Raman intensity.
Specialized immersion objectives replace the intervening space (air) between the objective and the sample using a material that matches the index of refraction of the sample, thus removing the spherical aberration and recovering the loss of spatial resolution and intensity. Oil immersion objectives typically use oils that have an index of refraction around 1.5. Using oil as the intermediate material between the objective and the sample also allows for a higher numerical aperture that helps increase the Raman intensity.
To learn how 3-D Raman imaging is enhanced with oil immersion objectives, read your newly published application note: “Chemical information from subsurface structures with 3-D Raman imaging and oil immersion objectives.” Download “Chemical Information from, Subsurface Structures with 3-D Raman Imaging and Oil Immersion Objectives.”
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