Analytical Technologies Singapore

What is a Confocal Raman microscopy?

What is a confocal Raman microscope?

A Raman microscope is a combination of a Raman spectrometer with a standard microscope, allowing high magnification visualisation of a sample and Raman analysis with a microscopic laser spot. 

However, just adding a microscope to a Raman spectrometer does not give a controlled sampling volume – for this a spatial filter is required.  Confocal Raman microscopy refers to the ability to spatially filter the analysis volume of the sample, in the XY (lateral) and Z (depth) axes.
 
The Raman microscope allows the Raman microscopy to be performed with microscopic spatial resolution:
  • Analysis and identification of individual particles with dimensions down to 0.5 µm.
  • Characterisation of sample features with dimensions down to 0.5 µm.
  • Location and identification of microscopic contaminants.
  • Raman mapping (imaging) of sample features to show the distribution of components, with a spatial resolution down to 0.5 µm.

Are all Raman microscopes confocal?

Some Raman microscopes do not have confocal optics. Adding a microscope only assists in giving lateral (XY) spatial resolution, but does not provide depth (Z) spatial resolution. For this confocal optics are required. Several methods are in use today (i.e. true confocal aperture, or pseudo confocal slit-binning techniques) with some are better than others. However, only by using a true confocal Raman microscope, it is then possible to analyse individual particles or layers with dimensions as low as 1 µm and below. 

For a true confocal design, the limits of spatial resolution are defined principally by the laser wavelength and quality of the laser beam used, and the type of microscope objective selected and so on. For the highest spatial resolution, a correctly matched high magnification objective and visible laser excitation will often produce optimum results. Typical spatial resolution is in the order of 0.5-1 µm.

Can a Raman microscope be used for depth profiling & analysis of features below the surface?

Yes.  A confocal Raman microscope can be used to analyse features below the sample surface provided the sample matrix is transparent to the laser.  Typical examples of such analyses include fluid/gas inclusions, contaminants in glass, and layered polymer structures.

On a basic system, manual focusing requires locating the required position within the sample, followed by spectral analysis.  If the Raman microscope is equipped with motorised Z (focus) control, then it is possible to acquire depth (Z) profiles through the sample automatically. 

Such a profile comprises a full Raman spectrum at each and every depth within the profile, and is then interrogated to generate intensity profiles based on material composition and structure:

  • Raman peak intensity yields a profile of material concentration and distribution
  • Raman peak position yields a profile of molecular structure and phase and material stress/strain
  • Raman peak width yields a profile of crystallinity and phase
  • A system which has additional XY motorised sample control can be used to optically slice through the sample, for example, to create an XZ or YZ Raman spectral image.


A system which has additional XY motorised sample control can be used to optically slice through the sample, for example, to create an XZ or YZ Raman spectral image.

Confocal Raman microscopy with other techniques

​Confocal Raman microscopy can be combined with numerous other techniques to attains a more comprehensive understanding of the sample.

1. TIP ENHANCED RAMAN SCATTERING, OR TERS

TERS (Tip Enhanced Raman Spectroscopy) brings Raman spectroscopy into nanoscale resolution Imaging. TERS is a super-resolution chemical technique. Better yet, it is a label- free super-resolution imaging technique which has been extended by our novel technology into an important new imaging technology.

TERS imaging is performed with an AFM/Raman system, where a Scanning Probe microscope (SPM that can be used in atomic force, scanning tunneling, or normal/shear force mode) is integrated with a confocal Raman spectrometer through an opto-mechanical coupling. The scanning probe microscope allows for nanoscale imaging, the optical coupling brings the excitation laser to the functionalized tip (or probe), and the spectrometer analyzes the Raman (or otherwise scattered) light providing a hyperspectral image with nanometer scale chemical contrast.

2. TRANSMISSION RAMAN SPECTROSCOPY, OR TR

Transmission Raman Spectroscopy (or TRS) is a form of Raman analysis which is ideally suited for bulk analysis of opaque/turbid materials. Transmission Raman is based on the collection of Raman light propagating through the sample in the direction of the excitation laser – in essence, the sample is illuminated with the excitation laser from one side, and the Raman signal is collected from the other.

Despite the sample being opaque, light from the laser can pass through the sample via light scattering processes. Many of these photons contain Raman information, and thus Transmission Raman spectroscopy is possible.

Unlike traditional Raman spectrometers and microscope systems, the transmission geometry allows true bulk analysis from the entire volume of the sample (for example, a pharmaceutical tablet).

Transmission Raman is non-contact, non-invasive and non-destructive. It requires no sample preparation. Importantly, the measurement is insensitive to particle size effects, sample homogeneity and orientation.
Transmission Raman spectroscopy can be used to understand:
  • API concentration
  • Content uniformity
  • Polymorphism
  • Crystallinity
  • Powder composition and purity
  • Solid form

3. ​RESONANCE RAMAN SPECTROSCOPY

Resonance Raman spectroscopy is a variant of ‘normal’ Raman spectroscopy. ‘Normal’ Raman spectroscopy uses laser excitation at any wavelength to measure the Raman scattering of this laser light.

Notwithstanding the many practical issues caused by the use of different laser wavelengths, the end result will be very similar to whatever wavelength is used.

In resonance Raman, the excitation wavelength is carefully chosen to overlap with (or be very close to) an electronic transition – this typically means in an area of UV-visible absorption. Such overlap can result in scattering intensities which are increased by factors of 102-106 – thus, detection limits and measurement times can be significantly improved. However, since the excitation coincides with UV-visible absorption, fluorescence backgrounds can be significant and more problematic than with ‘normal’ Raman scattering.

An alternative approach is Surface Enhanced Raman Scattering (SERS), which offers a similar order of magnitude increases in intensity. The advantage of SERS over resonance Raman is that fluorescence is suppressed while the Raman is enhanced, thus removing the fluorescence background problem of resonance Raman.

For certain specific applications, the benefits of resonance Raman can be powerful. One such example is the use of resonance Raman for the analysis of environmental pollutants, where concentrations in the parts per billion (ppb) and parts per million (ppm) range can be detected.

Practically, resonance Raman can be explored on any Raman system, and the actual measurement is made in the standard way. The obvious requirement is to have suitable laser excitation in order to meet resonance conditions.

Find out more about our Raman Spectrometers & microscopes here.

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