Label-free chemical imaging

Non-destructive label-free chemical imaging:

Raman and non-linear optical spectroscopy

Raman scattering can be used to measure and map chemical distribution within samples based on molecular vibrations.  The energy required to excite molecular vibrations within molecules depends upon the masses of atoms, the types of bonds and the chemical environment. Therefore, the scattering of incident monochromatic radiation generates frequencies reporting on the chemical functional groups present.

Spontaneous Raman spectroscopy techniques (both confocal and transmission) and coherent Raman techniques such as stimulated Raman scattering (SRS) microscopy are available at NPL and are suitable for ambient, non-destructive analysis of a range of sample types with minimal requirements for sample preparation. Both spontaneous and stimulated Raman scattering generate signals which are linear with concentration, enabling some degree of quantitative analysis. 

Confocal Raman microscopy

• Renishaw InVia Qontor Raman microscope
• Up to sub-micron spatial resolution
• Visible and NIR laser excitation sources
• Temperature-controlled stage and environmental chamber for temperature-controlled/dependent studies and cell imaging

Example applications:

• Surface chemistry of coatings
• Identification of crystalline phases and defects
• Characterisation of pharmaceutical and consumer health products
• Drug and chemical distribution in cells and tissues

Figure 1: Confocal Raman spectrum of Paracetamol, and confocal Raman maps of pharmaceutical tablets to reveal distribution of ingredients as labelled.  

Stimulated Raman scattering (SRS) microscopy at NPL

In SRS, two lasers are used, where the frequency difference is tuned to match the vibrational frequency of a molecule of interest (frequency can be identified in advance using confocal Raman spectroscopy). This stimulates the specific vibrational mode targeted and allows rapid label-free 3D imaging. The lasers can be raster scanned across the sample for imaging at speeds approaching video-rate. SRS images are comparatively free from non-resonant background such as emitted fluorescence, and the signal is linear with concentration. 

• Leica SP8 microscope with PicoEmerald-S laser system
• Sub-micron spatial resolution
• Second harmonic generation and emitted fluorescence signals can also be simultaneously detected in a separate channel (e.g. for label-free imaging of connective tissues).
• Environmental sample chamber for temperature-controlled studies

Example applications:

• Visualisation of ingredients within formulated products such as creams pastes and gels.
• Fate of topically applied drugs/chemicals
• Drug/chemical distribution in biological tissues
• Controlled release studies

Figure 2: SRS microscopy images: Left: Aqueous skin cream formulation; Middle: SRS and SHG composite image of skin treated with 4-cyanophenol *Data acquired in collaboration with the group of Prof. Richard Guy, University of Bath, UK. Funding for this research was made possible, in part, by the US FDA through grant 1U01FD006533-01. Right: SRS and SHG composite image of a Caterpillar.

For practical tips on how to achieve quantitative and reproducible measurements with stimulated Raman scattering microscopy, please refer to a recent tutorial review article published in the Analyst.

For further information on the use of SRS microscopy to assess formulated products, a case study can be found here.

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