Raman microscopy for advanced analysis

7th June 2019

The WITec alpha300 Ri inverted confocal Raman microscope
Fig.1. Particles in a cosmetic peeling cream sample
Fig.2. Raman flouresence image of a live cell culture of eukaryotic cells

Damon Strom provides a description of Raman microscopy and how it can be used to investigate particulate and biological samples

Raman microscopy is a fast and non-destructive chemical characterisation technique. It is based on the Raman Effect, which was discovered more than 90 years ago by Chandrasekhara Venkata Raman and documented in his paper A New Type of Secondary Radiation. Raman was later awarded the Nobel Prize in Physics for his work.

The Raman Effect results from the inelastic scattering of excitation light by the molecules of gaseous, liquid or solid materials. The interaction of a molecule with photons causes vibrations of its chemical bonds, leading to specific energy shifts in the scattered light that can be identified in its Raman spectrum. Any given chemical compound can be easily identified by this individual spectral ‘fingerprint’.

Less than one Raman-shifted photon is usually produced from one million incident photons. Therefore, the instruments used to acquire Raman signals should have optimised optical components, including highly sensitive detectors with low noise, and a confocal beam path to reject light from outside the focal plane and increase the signal-to-noise ratio. This optimisation enables the detection of Raman signals from even weak Raman scatterers and
extremely low material concentrations or volumes. Measurements carried out on delicate samples also benefit from the ability to acquire Raman spectra using very low excitation

Raman particle identification and characterisation

High-resolution investigations of particles are of great interest in fields of application such as environmental science, microplastics research, pharmaceutical research, medical device development, life sciences and many others. Combining a particle analysis tool with Raman microscopy makes it possible to find, classify and identify particles in a sample according to their size, shape and chemical characteristics. The physical and molecular attributes of the particles in a sample may be correlated and qualitatively and quantitatively evaluated.

The following measurement was performed with the WITec ParticleScout particle analysis tool in conjunction with a WITec alpha300 R confocal Raman microscope.

Fig. 1 shows the results of particle analysis carried out on a cosmetic peeling cream sample. Fig. 1A shows the overlay of an optical bright field microscope image with the corresponding confocal Raman image. Particles are identified according to their size and shape and further characterised by their molecular properties through confocal Raman imaging. The chemical analysis revealed anatase and boron nitride particles in an oil matrix (Raman spectra shown in Fig. 1B).

Further evaluation of the results determines the quantitative prevalence of the molecular sample components in the particles (Fig. 1C) and also the distribution of chemical compounds correlated to particle size (Fig. 1D). In extended analyses the chemical characteristics of particles could also be linked to parameters such as area, perimeter, bounding box, Feret diameter, aspect ratio, equivalent diameter and many others.

Raman and fluorescence

Fluorescence microscopy has been a widespread imaging method for the analysis of biological cells and organisms for decades. Samples are stained with fluorescent dyes or organisms are genetically engineered to express fluorescent proteins. The fluorescence signal is usually much stronger than the Raman signal. Nevertheless, correlative Raman fluorescence measurements are possible with an appropriate system.

The investigation described below was carried out using a WITec alpha300 Ri inverted confocal Raman imaging microscope to examine the cells in their aqueous cell culture medium in the Petri dish.

Fig. 2 shows a Raman fluorescence image of a live cell culture of eukaryotic cells. The cell nuclei were stained with the fluorescent dye DAPI. An excitation wavelength of 532nm was used for the Raman measurement. An image with 50 x 40 µm² and 150 x 120 pixels was acquired. A Raman spectrum was recorded at each pixel. In the correlative Raman fluorescence image, the nuclei are shown in blue (recorded with fluorescence microscopy), the nucleoli in green and the endoplasmic reticula in red (recorded with Raman microscopy). The corresponding Raman spectra are shown in the same colours.


Raman microscopy is a versatile and powerful tool for identifying the chemical components present in a sample. The speed and precision it offers, along with its ease of use and the minimal sample preparation it requires, makes it appealing to researchers in many fields. A Raman microscope of modular design that permits combinations with specialised software tools and other techniques extends its analytical range and utility.

Damon Strom is with WITec





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