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

Zoladek, Alina Bogumila; Johal, Ramneek Kaur; Garcia-Nieto, Samuel; Pascut, Flavius; Shakesheff, Kevin M.; Ghaemmaghami, Amir M.; Notingher, Ioan

Abstract

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. © 2010 The Royal Society of Chemistry.

Citation

Zoladek, A. B., Johal, R. K., Garcia-Nieto, S., Pascut, F., Shakesheff, K. M., Ghaemmaghami, A. M., & Notingher, I. (2010, June). Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy. Presented at SPEC 2010 Shedding Light on Disease: Optical Diagnosis for the New Millennium, Manchester, UK

• Presentation Conference Type: Conference Paper (published)
• Conference Name: SPEC 2010 Shedding Light on Disease: Optical Diagnosis for the New Millennium
• Start Date: Jun 26, 2010
• End Date: Jul 1, 2010
• Acceptance Date: Oct 5, 2010
• Online Publication Date: Oct 18, 2010
• Publication Date: Dec 1, 2010
• Deposit Date: Jan 3, 2023
• Journal: Analyst
• Print ISSN: 0003-2654
• Electronic ISSN: 1364-5528
• Publisher: Royal Society of Chemistry
• Peer Reviewed: Peer Reviewed
• Volume: 135
• Issue: 12
• Pages: 3205-3212
• DOI: https://doi.org/10.1039/c0an00508h
• Public URL: https://nottingham-repository.worktribe.com/output/3097551
• Publisher URL: https://pubs.rsc.org/en/content/articlelanding/2010/AN/c0an00508h