White Paper: Medical

Fundamentals of Confocal Raman Microscopy

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Microanalysis using Raman spectroscopy is an invaluable tool for providing high-resolution images of complex molecular samples. Since the Raman effect results from inelastically scattered laser light, this technique requires no labeling or tagging, unlike fluorescence microscopy. Additionally, because of the wavelength independence of the technique, visible laser light can be used, providing exceptionally high resolution when compared to other label-free molecular microscopies such as infrared.

As a result, Raman microscopy is now commonly used in a wide range of application spaces, including material science, pharmaceuticals, and biomedical. Unfortunately, despite its popularity in the spectroscopy community, the terms “Raman Microscopy” and “Confocal Raman” are used interchangeably, which often leads to a lot of confusion and miscommunication.

While it is true that confocal Raman microscopy is a subset of Raman microscopy, the unique optical geometry utilized in a confocal microscope presents both advantages and disadvantages not seen in traditional microscopy; for example, confocal Raman microscopy offers far superior spatial resolution than conventional Raman microscopy but this comes at the cost of collection efficiency.

All lasers used in Raman spectroscopy must have an extremely stable output spectrum since the Raman spectra are based on the shift in wavelength, not the absolute wavelength of the scattered light. While this is true in all Raman spectroscopy systems, this is particularly important for confocal Raman microscopes because the spectral resolution tends to be much finer than most portable or benchtop Raman spectrometers.

This white paper reviews the fundamental optical design of a confocal Raman microscope, discusses the advantages of this design for collecting Raman spectra, and explores the laser requirements and challenges facilitated by this geometry.

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