Optics 101

What is Raman Microscopy?

​Raman spectroscopy is a technique used in chemistry to determine which molecules are present in a sample and the bonds between them, resulting in a structural fingerprint by which said molecules can be recognized.

Raman spectroscopy uses light from a laser – in either the visible, near infrared or near ultraviolet range – to observe vibrational and other low-frequency modes of molecules as a means of determining which functional groups, and therefore molecules, are present in a sample.

Understanding how Raman Spectroscopy Works

When light is focused on a sample, photons interact with the molecules and they are then either reflected, absorbed, transmitted or scattered. Raman spectroscopy studies the scattering of photons, specifically the inelastic scattering of a photon, which causes a change in its wavelength.

The majority of photons interacting with molecules scatter elastically – this is known as Rayleigh scattering, and the photons have the same wavelength as the incident light (the laser). However, around one in a million photons are scattered inelastically and its wavelength is shifted higher or lower than the incident light – this is known as the Raman effect, named after Sir C V Raman who discovered it using sunlight in 1928.

The Raman effect involves the interaction of photons with the electron clouds of different functional groups on molecules, this leads to electronic transitions to virtual states which results in photon leading to the photons losing or gaining energy. This interaction involves polarization of the electron cloud, so the more polarizable a functional group is the greater the interaction strength, and therefore the greater the energy change. This change in energy can be directly related to specific functional groups and therefore overall molecular structure.

The shift in photon energy due to the Raman effect is observed on a spectrum as two small peaks of equal size either side of the main (elastic) absorption. This corresponds to the photons which have lost or gained energy inelasticly, these lines are called the Stokes and Anti-Stokes lines respectively.

A diagram illustrating the electronic transitions which occur during Raman (inelastic) scattering. Rayleigh (elastic) scattering  is shown as a comparison | wikimedia

Raman spectroscopy is not easy - spontaneous Raman scattering is often weak and it can be hard to separate the inelastically scattered light from the elastic scattered light. Although the technique was used to provide the first catalog of molecular vibration frequencies, it fell out of favour when commercial infrared spectrophotometers emerged in the 1940s. The introduction of the laser in the 1960s, however, simplified Raman spectroscopy instruments and boosted the sensitivity of the technique.

Over the years, many advanced types of Raman spectroscopy have been developed to enhance sensitivity or spatial resolution including surface-enhanced Raman, tip-enhanced Raman and polarized Raman. It has also been incorporated into other techniques, including in microscopic analysis.

Raman Microscopy

Raman microscopy allows the user to obtain Raman spectra of minute samples or microscopic areas of larger samples. It utilizes a Raman micro-spectrometer - a specially designed Raman spectrometer integrated with an optical microscope - rather than the standard instrument, and there are variations depending on the type of microscopy required, eg confocal.

In direct imaging, the whole field of view is studied for scattering over a small range of wavenumbers, while in hyperspectral or chemical imaging, thousands of Raman spectra are obtained from all over the field of view which are used to generate images showing the location and number of different components.

It is ideal for use where little sample is available, or to enhance certain effects over local regions. The technique is not affected by water – which can interfere with other spectral analyses – and so is useful for examining minerals, polymers and ceramics, cells, proteins, organs and forensic trace evidence.

As reported in Proceedings of the National Academy of Sciences of the United States of America, the technique has been used to identify anthrax endospores inside sealed envelopes. In the art world, it has been used to determine the authenticity of Leonardo Da Vinci’s Salvator Mundi by analyzing the dyes and pigments used: a copy of his painting was proven to be his own work.

The controversial 'Salvator Mundi', by Leonardo Da Vinci, was authenticated using Raman micro-spectrosocpy | wikimedia

In microbiology, a variation called confocal Raman microscopy has been used to map intracellular distributions of macromolecules – proteins, polysaccharides and nucleic acids – in bacteria and microalgae, while in medicine, it has been shown to be able to detect slight biochemical changes within cells associated with the onset of cancer.

References and Further Reading

  1. http://www.microspectra.com/support/the-science/raman-science
  2. https://en.wikipedia.org/wiki/Raman_spectroscopy#Microspectroscopy
  3. https://www.forensicmag.com/article/2013/02/utilization-infrared-and-raman-micro-spectroscopy-chemical-analysis-art
  4. https://agoracienciasblog.files.wordpress.com/2018/03/ejemplo-pc3b3ster-1.pdf

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