Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy

• Published in: Analyst (London) 2010-12, Vol.135 (12), p.3205-3212
• Main Authors: BOGUMILA ZOLADEK, Alina, RAMNEEK KAUR JOHAL, GARCIA, Samuel, PASCUT, Flavius, SHAKESHEFF, Kevin M, GHAEMMAGHAMI, Amir M, NOTINGHER, Ioan
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Analytical chemistry; Applied sciences; Cells, Cultured; Chemistry; Cluster Analysis; Coculture Techniques; Dendritic Cells - chemistry; Dendritic Cells - ultrastructure; Exact sciences and technology; General, instrumentation; Global environmental pollution; Humans; Immunological Synapses - chemistry; Immunological Synapses - ultrastructure; Laminin - chemistry; Microscopy, Confocal - methods; Pollution; Spectrometric and optical methods; Spectrum Analysis, Raman - methods; T-Lymphocytes - chemistry; T-Lymphocytes - ultrastructure
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c0an00508h

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.