Journal
ANALYST
Volume 135, Issue 12, Pages 3205-3212Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c0an00508h
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Funding
- Biotechnology and Biological Sciences Research Council UK [BB/G010285/1, BB/F001142/1]
- BBSRC [BB/F001142/1, BB/G010285/1] Funding Source: UKRI
- MRC [G0601750] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/G010285/1, BB/F001142/1] Funding Source: researchfish
- Medical Research Council [G0601750] Funding Source: researchfish
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Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.
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