SPECTROSCOPY 47
Te application of correlative Raman-AFM microscopy for bioimaging has been elegantly presented by studies of murine blood vessels and atherosclerotic plaques therein. Molecules such as cholesterol and its esters, elastin and heme, were localised, while AFM imaging added topographical information about the vessel’s structure6, 7
.
Correlative Raman imaging and scanning electron (RISE) microscopy is a powerful new analytical method for the analysis and interrelation of the structural properties and molecular composition of samples8
. With
a hybrid system consisting of a Raman microscope and an SEM the sample remains within the vacuum chamber and is automatically transferred from one measurement position to the other. Here, RISE microscopy was performed on hamster brain tissue (see Fig. 2). In the SEM image the white
REFERENCES: 1
and grey brain matter can be distinguished by their structural differences. Overlaying the SEM with the colour-coded Raman image generated from the Raman spectra reveals a more detailed view of the distribution of the grey and white matter. Both tissues can be clearly differentiated by their characteristic Raman spectra. While the utility of confocal Raman imaging as a stand-alone technique or in combination with AFM or SEM has been ably demonstrated by the study shown above and many others, we are sure that it will fulfil its promise for life sciences and biomedical applications in the near future.
For more information ✔ at
www.scientistlive.com/eurolab
Karin Hollricher is with WITec.
www.witec.de
T. Dieing, O. Hollricher, J. Toporski, Confocal Raman
Microscoopy. W. Rhodes, Ed., Optical Sciences (Springer, Heidelberg Dordrecht, London New York, 2010), vol. 158. 2
A. Hermelink, A. Brauer, P. Lasch, D. Naumann, Phenotypic
Fig. 2. RISE image of a hamster brain tissue sample. In the colour-coded Raman image the white brain matter is shown in green, the grey brain matter in red. The corresponding Raman spectra reveal the different spectral characteristics of the brain tissue. Image parameters: 100 μm x 100 μm, 300 x 300 pixels = 90,000 spectra, integration time 50 ms/spectrum
receptor EGFR (epidermal growth factor receptor). As clinical studies have indicated that colon cancer cells can acquire resistance to antibody- based therapy through mutation, this imaging technique could be used to follow the response to therapy.
Correlative approaches involving Raman microscopy For many endeavours, knowledge not only of the
sample’s chemical composition but also of its morphology on a sub-micrometre scale is crucial. Such information can be gained by correlative microscopy combining Raman imaging with either atomic force microscopy (AFM), scanning near-field optical microscopy (SNOM) or scanning electron microscopy (SEM). Having two or more technologies integrated into one instrument greatly streamlines correlative analyses.
heterogeneity within microbial populations at the single-cell level investigated by confocal Raman microspectroscopy. Analyst 134, 1149-1153 (2009). 3
H. Abramczyk et al., The role of lipid droplets and adipocytes
in cancer. Raman imaging of cell cultures: MCF10A, MCF7, and MDA-MB-231 compared to adipocytes in cancerous human breast tissue. Analyst 140, 2224-2235 (2015). 4
S. F. El-Mashtoly et al., Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy. Analyst 139, 1155-1161 (2014). 5
H. K. Yosef et al., In vitro prediction of the efficacy of molecularly targeted cancer therapy by Raman spectral imaging Anal. Bioanal. Chem. 407, 8321-8331, (2015). 6
M. Pilarczyk et al., Multi-methodological insight into the
vessel wall cross-section: Raman and AFM imaging combined with immunohistochemical staining. Biomed Spectroscopy and Imaging 2, 191-197 (2013). 7
K. M. Marzec et al., Visualization of the biochemical markers of atherosclerotic plaque with the use of Raman, IR and AFM. J. Biophotonics 7, 744-756 (2014). 8
O. Hollricher, Supercharging correlative microscopy. Anal. Scientist, 52 - 54 (2015).
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