The Science of Micro Raman Spectroscopy

Raman Spectroscopy

When electromagnetic energy undergoes interaction with a material, it can be absorbed, reflected, scattered, or transmitted. One kind of scattering is Raman scattering, which serves as the base for Raman spectroscopy. Raman scattered light provides data of the vibrational modes of the samples due to which Raman spectroscopy is beneficial, offering Raman spectrum. Similar to IR spectroscopy, Raman spectroscopy imaging entitles the identification of molecules and their functional groups. This article deals with a variant of Raman spectroscopy, which is micro Raman spectroscopy. Information and science about micro Raman spectroscopy have been briefed.

Micro Raman Spectroscopy

In micro Raman spectroscopy, a Raman microspectrometer is employed in the place of a conventional Raman spectrometer. A Raman microspectrometer contains a specifically designed Raman spectrometer, which is amalgamated with an optical microscope. This facility permits the technician to acquire Raman spectra of microscopic samples and microscopic areas of macroscopic samples.

Advantages of Micro Raman Spectroscopy

The pros of using micro Raman spectroscopy are:

• Much lesser amount of samples is enough for analysis
• Certain effects are enhanced in very localized regions.

Design of Micro Raman Spectrometer

For obtaining different information about the sample from the Raman scattering, micro Raman spectrometer is able to be configured with different colored lasers.

A Micro Raman spectrometer is also referred to as a Raman microspectrometer. It combines both the aspects of Raman spectrometer and microscope. It is purposely built equipment that can do Raman spectroscopy and digital imaging.

A microscope, when used individually, is optical equipment that uses mirrors and lens for the production of magnified images of microscopic objects or microscopic areas of macroscopic objects. The sample substance is illuminated through the objective, with a laser consisting of a very narrow wavelength range.

Gathering the Raman scattered light from the sample is the objective of the microscope. The light is concentrated and frames an image on the entrance aperture of the Raman spectrometer.

The Raman spectrometer portion is a kind of optical instrument utilized for the estimation of the intensity of light relative to its Stokes shift from the wavelength of the exciting laser light. The shit is provided in wavenumbers. A beam of light is collected from a sample, which enters the device, and diffraction grating separates the Stokes shifted frequencies. The separated light is concentrated onto a CCD array detector, in which the intensity of each frequency is estimated by an individual pixel on the array. 

Then, the CCD is read-off to a computer monitor and the result obtained is a spectrum, which reveals the intensity of inelastically scattered light versus wavenumbers in relation to the wavelength number of the laser used for excitation.

In a micro Raman spectrometer, a Raman spectrometer is incorporated with a specially designed microscope. The Raman spectrometer is built into a microscope with a digital imaging system, which gives rise to the collection of the maximum amount of light from the smallest samples. In addition, they are very flexible instruments for the measurement of Raman spectra of microscopic areas.

Applications

Micro Raman spectroscopy is used for the identification of different molecules and functional groups present within larger molecules. Bonds formed between the atoms possess specific vibrational frequencies, which correspond to the masses of atoms and the potency of the bond between them. The complex molecules display many peaks and can be promptly recognized by the fingerprint or pattern created by the peaks. There are plenty of applications of micro Raman spectrometers as they are able to recognize microscopic samples or microscopic areas of bigger samples in a non-destructive way.

Forensics

• Plastics & polymers
• Drugs of abuse
• Paint chips
• Ink
• Explosives

Semiconductor

• Identification of contaminants
• Materials research
• Development of OLED
• Diamond-like films
• Silicon crystal state
• Silicon strain
• Thin film quality control

Art

• Identification of pigment on paintings
• Recognition of dye on textiles

Geology

• Geological research
• Analysis of petroleum
• Authentication of gemstone

Materials Science

• Diamond films
• Carbon nanotubes

Pharmaceutical

• Discovery of drugs
• Identification of contaminants
• Development of microfluidic devices
• Counterfeit pharmaceutical recognition

Miscellaneous

• Quality control of polymers
• Dye development
• Development of pigments

Micro Raman spectroscopy is perceived as a conventional Raman technique merely with superior resolution, which is bounded by a far-field diffraction limit.

Micro Raman spectroscopy is also known as Raman microscopy. It embraces the pairing of a conventional Raman spectrometer with an optical microscope. This conjugation delivers immense power in sampling portions with higher spatial resolution in the order of 1 micrometer. 

Types of Lasers used

For a Raman microscopy set up, the following laser sources can be used:

• Continuous wave lasers - Helium-neon (632.8 nm), krypton (337.4-676.4 nm), argon (351.1 - 514.5 nm).
• Diode lasers - 785 nm, 810 nm, 830 nm.
• Pulsed laser - Nd:YAG 1064 nm

Design of Micro Raman Spectrometer

• The monochromator is utilized to disperse the collected radiation onto the multichannel detector. CCD or Intensified diode arrays are the most commonly used detectors of Raman microscopy for simultaneous detection of a large spectral range.
• Raman spectroscopy and microscopy are both powerful on their own, but when blended, they put forward the possibility to chemically investigate the smallest objects (> 0.5 micrometers), and consequently, they relate spectral with spatial data.
• On the contrary with IR spectroscopy, Raman microscopes are easily imploded as they utilize light, which is compatible with simple glass optics. Hence, Raman microscopes are regularly created on the grounds of a very high optical microscope. 
• There is no elaborate sample preparation for micro Raman spectroscopy. Samples are placed under the microscope as they are. Preparation of cross sections and cutting of large workpieces to sizes fit on the stage is at most done as preparative steps.
• The sample may not show strong absorbance or fluorescence of the excitation wavelength as occurring in Raman spectroscopy. Few samples need a confocal Raman microscope, which provides spatial resolution in three dimensions. Through this approach, it is probable for measurement inside containers like glass vials, thereby characterizing samples in 3D.

Calibration

For obtaining reliable and precise results of the sample, the calibration of the wavelength axis is a requisite. Several operational changes in Raman microscopy generally possess less or more severe consequences in terms of calibration of wavelength. Though modern microscopes provide continuous calibration for maximum convenience, recalibration is done by estimating a silicon standard. If the equipment is not calibrated continuously, recalibration should be done routinely. Minor adjustments like aperture, grating, mirror, vibrations, sudden shocks, variations, temperature shifts are performed to ensure delivery of optimal spectral data.  

Conclusion

Initially, a short introduction to Raman spectroscopy and micro Raman spectroscopy has been provided. The pros of micro Raman spectroscopy have been discussed. A way of designing a micro Raman spectrometer has been given. Then, applications of micro Raman spectroscopy in different fields have been listed. Laser usage in Raman microscopy has been explained. Subsequently, calibration of Raman microscopy has been detailed.