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Getting Started with Raman Spectroscopy: What You Need to Know

Are you an assiduous biologist who prefers label-free imaging methods for biological samples analysis? Raman spectroscopy offers you a wonderland of imaging technique with unlimited benefits.

To start with, Raman Spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light usually from a laser in the visible or near infra-red part of electromagnetic spectrum. It probes vibrational modes of a molecule and crystals of solid, liquid and gaseous samples. It is commonly used in living cells and tissues to provide a fingerprint by which molecules can be identified.

Potential use

Laser-based Raman spectroscopy can be used to detect all your biological samples as every molecule or chemical species has its own unique characteristic Raman spectrum.

The benefits of Raman spectroscopy include:

  • It is a non-destructive scattering technique.
  • It can be used on live samples. You do not need to fix or section your specimen
  • Raman spectra analysis requires extremely small volume (< 1 µm in diameter) sample collection for species identification.
  • The technique is insensitive to aqueous absorption bands. Thus, Raman spectroscopy is suitable for your microscopic examination of cells and proteins.
  • Raman spectroscopy is quick. A typical analysis requires only a few seconds.
  • Raman technique is highly selective. You can use it to differentiate between molecules in biological species that are very similar, for example breast cancerous cells variants.

Essential components

You might think that such a powerful technique would require a multitude of components. But fear not, you can set up a Raman system in a laboratory using only four main components, namely:

1. Excitation source (laser)

A good Raman laser should feature a number of different characteristics including narrow linewidth, small form factor, low power consumption and extremely stable power output with stable wavelength output.

2.  Sample interface

The second component is sample interface. A commonly used interface in Raman spectrometry is the fiber-optic probe, which has the important feature of high optical Raman density cut-off. Due to its flexibility, it can be easily adapted into a variety of sample chambers such as liquid and gas flow cells, gas flow cells as well as and optical microscope.

3.  Spectrometer (filter or wavelength selector)

A spectrometer, such as a notch filter, is used to select and obtain the Raman spectrum of the sample. The major performance factors are high resolution, low noise, small form factor, and low power consumption.

4.  Detector

Choose the detector to match the excitation laser employed. For ultraviolet excitation, a photomultiplier tube (PMT) or charged coupled detector is normally the best choice, whereas a standard charged coupled detector is typically used for visible excitation and an indium gallium arsenide array for near infrared.

Limitations

Although Raman spectroscopy is powerful, it does have some limitations. In particular, spontaneous Raman scattering is typically very weak. Therefore, the main difficulty of Raman spectroscopy lies in separating the weak inelastically scattered light (the data you want to collect) from the intense Rayleigh scattered laser light (noise). More precisely, the major problem is not the Rayleigh scattering itself, but the fact that the intensity of stray light from the Rayleigh scattering may greatly exceed the intensity of the useful Raman signal in the close proximity to the laser wavelength.

To limit the noise in the system, cut off the spectral range close to the laser line where the stray light has the most prominent effect. You can buy commercially available interference (notch) filters which cut off the spectral range of ± 80-120 cm-1 from the laser line. This eliminates stray light, but does not allow the detection of low-frequency Raman modes in the range below 100 cm-1.

Hopefully I have lit the way for you to get started with Raman spectroscopy as an imaging technique.

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