APPLICATION NOTE

Correlative Confocal Raman Microscopy
A powerful tool for non-destructive, label-freechemical and structural characterization

7x7mm²WITec GmbH, Lise-Meitner-Str. 6, 89081 Ulm, Germany
Tel. +49 (0) 731 140 700, Fax +49 (0) 731 140 70 200
info@WITec.de, www.WITec.de

RISE microscopy image of graphene: SEM image
overlaid with a color-coded confocal Raman image

Topographic Raman microscopy
image of a pharmaceutical tablet

Confocal Raman microscopy is auniquely effective method for analyzing
the chemical composition of heterogeneous samples on the sub-micrometerscale.

Raman imaging of banana pulp: Anoverlay of a confocal Raman image ona white-light image. Green: starch; red:carotenoids; blue: waterapplication noteLise-Meitner-Str. 6, D-89081 Ulm, Germany
Tel. +49 (0) 731 140 700, Fax +49 (0) 731 140 70200
www.WITec.de, info@WITec.de

The Raman principle

Raman imaging

The Raman effect is based on theinelastic scattering of light by themolecules of gaseous, liquid or solidmaterials. The interaction of a molecule with photons causes vibrations ofits chemical bonds, leading to specificenergy shifts in the scattered light.
Thus, any given chemical compound
produces a particular Raman spectrum
when excited and can be easily identified by this individual “fingerprint.”

In Raman imaging, a confocal micro­
scope is combined with a spectrometerand a Raman spectrum is recorded atevery image pixel. The resulting Raman
image visualizes the distribution of thesample’s compounds. Due to the highconfocality of WITec Raman systems,
volume scans and 3D images can also
be generated.

Raman spectroscopy is a well-established, label-free and non-destructive
method for analyzing the molecularcomposition of a sample.

Technologies and Applications
Confocal Raman microscopy for 3D imaging
Volume scans and 3D images are valuable for providing
information about the dimensions of objects or the distribution of a certain compound throughout a sample. Here,a 3D Raman image of a pharmaceutical emulsion consisting of water (blue), oil (green) and some impurities (red) isshown. The oil was partially removed from the image for abetter sample overview.

40x40x15µm³

Raman - SEM (RISETM) imaging in geology
Geological samples are often very complex. With confocal
Raman imaging, the spatial distribution and association ofcomponents or mineral phases can be evaluated. In thisiron-rich sample, several crystal forms of hematite andgoethite were identified by their Raman spectra. From thesespectra, a Raman image was generated and overlaid witha scanning electron microscopy (SEM) image. Both measurements were performed with a combined Raman-SEM
system (RISE = Raman Imaging and Scanning Electron
microscopy).

Topographic Raman imaging
with TrueSurfaceTM

Correlative Raman-AFM
imagingWith WITec’s TrueSurface
microscopy option confocal
Raman imaging can beguided by the surface topo­
graphy of even rough, inclined
or irregularly-shaped samples.
A sensor actively monitors thesample surface and keeps it infocus. As examples, topographic Raman images of a pain
relief tablet (title page) and of
paper are shown.

Combining Raman imaging andatomic force microscopy (AFM) in
one instrument makes it possible
to obtain chemical and structural information from the same
sample area at the highest resolution. The AFM phase image of apolymer blend (top) was overlaid
with the Raman image. Thus, thesample’s molecular components
and its physical features could becorrelated (bottom).