Raman microspectrometry is a powerful non-destructive technique that can be used to assess the chemical composition of samples in real time.1 Raman microspectrometry is compatible with a number of sample types, from tissues to materials for electrodes.2
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Raman microspectrometry works by combining the spatial resolving power of microscopy methods with the spectroscopic information provided by Raman spectroscopy. By translating a focused beam of light over the sample of interest and collecting the Raman signal for analysis, a spatial map of the chemical composition of the sample can be constructed.
Traditional optical microscopes cannot provide the same chemical information without the use of specific fluorescent tags that only bind to certain sites or compounds. Raman microspectrometry often needs little to no sample preparation, even for biological cell and tissue samples, though the removal of some organic contaminants can be helpful when dealing with trace element analysis.3
Principles of Raman Spectroscopy
Raman spectroscopy is a quantitative and qualitative analysis technique that relies on the assignment of ‘chemical fingerprints’ to identify whole chemical compounds or the presence of particular functional groups.
In a Raman measurement, light of a given frequency interacts with the sample of interest. This incident radiation can then interact with the vibrational mode or electronic states in the sample and be scattered having gained or lost an amount of energy that is dependent on the state the radiation interacted with.
The vibrational frequency of a bond is dependent on the masses of the atoms involved in the bond, as well as the strengths of the bond between them. By mapping out the vibrational frequencies of all the Raman active modes in a sample, a unique fingerprint of the molecule can be constructed that can be used for identification by comparison to spectral databases or first principles assignments.
Raman spectroscopy is particularly successful at the identification of certain functional groups or bond stretching modes as these typically produce the most intense Raman signals. C=C and C=N stretches generally occur in the same frequency region, no matter what the molecule is, so can be very useful for assignment.
Instrumentation and Setup for Raman Microspectrometry
One of the biggest challenges in Raman spectroscopy and microspectrometry is the inherently weak nature of the Raman signal and the competition between the Raman signal and fluorescence background that plagues all Raman measurements.
The intensity of the Raman signal scales with the inverse of the fourth power of the wavelength, so the shorter the wavelength used, the more intense the Raman signals are. However, the shorter the wavelength used, the greater the probability of exciting an electronic transition that can lead to fluorescence from the sample, which causes a broad background that can obscure the Raman features of interest.
The use of high-intensity lasers for Raman excitation has the advantage of both high intensities for improving the signal levels and also the ease of focusing the beam to achieve good spatial resolution in the measurements.
A typical Raman microspectrometry instrument will have an excitation source, microscope optics for beam handling, a sample holder, often with a translation stage if wide-field illumination is not being used, and a Raman spectrometer for the detection of the scattered Raman radiation. Some instruments also make use of effects such as surface-enhanced Raman scattering (SERS) as a way of boosting the Raman signal levels.4
Applications of Raman Microspectrometry
Key applications of Raman microspectrometry are in the life sciences1, cultural heritage studies4 and in materials analysis.2,5 The high spatial resolution of Raman microspectrometry and the strong Raman signals given by solvents such as water means Raman microspectrometry is one of the few techniques that can be used to probe fluid inclusions in materials.5
Recently, Raman microspectrometry has been used as a tool for microparticle analysis, which includes the identification and classification of microplastics.6 The chemical information provided by the Raman spectra recorded can be used to identify what type of plastic has produced the particles and the microscopy element can be used for particle sizing.
The reason Raman microspectrometry works so well for this application is that most polymers have a well-known chemical fingerprint and individual analysis protocols do not need to be developed for each new microplastic, as they do for some chromatography methods. There are some size limitations to what can be studied, but Raman microspectrometry has proved versatile and broadly applicable for many sample types.
Advantages and Limitations of Raman Microspectrometry
Raman spectroscopy has been a very widely used technique in many industries, from oil and gas to pharmaceuticals and so there is extensive spectroscopic data available for compound identification which helps with the quantitative analysis of Raman microspectrometry. Advances in laser technologies and the availability of methods such as SERS in combination with microscopy are also helping to overcome some of the inherent limitations of the weak Raman signals, which are always a challenge when making any type of Raman measurements.
As either visible or near-infrared wavelengths are typically used for Raman excitation, the minimal spatial resolution achievable by Raman microspectrometry is in the hundreds of nanometers due to the diffraction limit of the wavelengths of light used. There are attempts to use image processing to improve the achievable resolution in measurement through post-processing of the data.7
See More: Raman Spectroscopy Mapping of Bilayer Graphene Stacks
References and Further Reading
- Lee, K. S., et al. (2021) Raman microspectroscopy for microbiology. Nature Reviews, 1, p. 80. doi.org/10.1038/s43586-021-00075-6
- Baddour-Hadjean, R. & Pereira-Ramos, J. (2010) Raman Microspectrometry Applied to the Study of Electrode Materials for Lithium Batteries. Chemical Reviews, 110, pp. 1278–1319. doi.org/10.1021/cr800344k
- Jolivet, A., et al. (2013) Preparation techniques alter the mineral and organic fractions of fish otoliths : insights using Raman. Analytical and Bioanalytical Chemistry, 405, pp. 4787–4798. doi.org/10.1007/s00216-013-6893-2
- Caggiani, M. C., & Colomban, P. (2019) Raman microspectroscopy for Cultural Heritage studies. Physical Sciences Reviews, 3(11), pp. 1–18. doi.org/10.1515/psr-2018-0007
- Burke, E. A. J. (2001) Raman microspectrometry of fluid inclusions: the daily practice. Lithos, 55, pp. 139–158. doi.org/10.1016/S0024-4937(00)00043-8
- Anger, P. M., et al. 2018) Raman microspectroscopy as a tool for microplastic particle analysis. TrAC – Trends in Analytical Chemistry, 109, pp. 214–226. doi.org/10.1016/j.trac.2018.10.010
- Cui, H., et al. 2016) Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration. Optics Express, 24(10), p. 10767. doi.org/10.1364/oe.24.010767
