46 SPECTROSCOPY
Illuminating Raman imaging S
Karin Hollricher on the potential of confocal Raman imaging in the life sciences sector
ince the invention of the microscope, imaging has continued to increase in sensitivity and is today a very important tool in analytical science generally and life sciences in particular. A more recent form of imaging is Raman microscopy, which during the past 15 years has led to the development of mature instruments that due to the molecular sensitivity of the technique are increasingly used to analyse prokaryotic and eukaryotic cells and tissues. Correlative approaches involving Raman microscopy and imaging modes capable of structurally characterising specimens have opened new avenues for the analysis of living cells on the sub-micrometre scale.
Raman imaging is a technique that can provide insight regarding chemical component distribution on a sample with a diffraction- limited spatial resolution of approximately λ/2 of the
excitation wavelength, down to 200nm1
. A Raman image can
be compiled from the spectral data at each measurement point of a sample. Confocal Raman microscopy systems also allow for the analysis of samples along the vertical Z-axis. In this way, 3D-images and depth profiles can be generated. Te technique is non-invasive, non-destructive and requires minimal, if any, sample preparation. Importantly for applications in the life sciences, Raman imaging is fairly insensitive to water. Te Raman imaging results shown in the following were obtained with WITec microscopes. RISE microscopy was carried out with a WITec Raman/Tescan Scanning Electron Microscope combination.
Raman imaging of cells and tissues Te detection and
characterisation of bacterial species is central to medical diagnostics and food analysis, but also to environmental applications. Raman spectroscopy has gained momentum as a tool for the identification of microorganisms by their individual Raman spectra resulting from minute differences in their biochemical composition. It has also been shown that based on specific
stretching signals, different phenotypes of the same bacterial species could be differentiated2
.
For example, the chain-forming cells of the rod-shaped Bacillus cereus could be visualised by the C-H stretching Raman signal that is typical for organic compounds (see Fig. 1). Te bacterial endospores still inside the vegetative cells were recognised by their high pyridine content while Poly-ß- hydroxybutyric acid (PHB), an energy storage compound, was identified from its C=0 ester stretching band in the spectrum. Recent reports have explored the potential of Raman imaging to detect discrepancies in the molecular composition of healthy and cancerous cells. For example, benign cells, mildly malignant and highly malignant breast cells were shown to differ in the amount and the content of lipid droplets3
. Even the cells’
responses to drug treatments could be visualised with Raman imaging. In a study on colon cancer cells the uptake of the drug erlotinib was followed by the increase of particular Raman signals in the cell4
. Additional
experiments revealed that the anti-cancer antibodies induce prominent changes in the Raman spectra of the cells5
, for example
a reduction of lipid droplets. Te biologicals target the cell surface
Fig. 1. Raman image of Bacillus cereus. Vegetative cells (yellow), endospores (violet)
and PHB (green) Image courtesy of Antje Hermelink, Robert-Koch-Institute, Berlin, Germany
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