Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy.
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.
School of Physics and Astronomy, University of Nottingham, Nottingham, UK.
Alina Bogumila
AB
Zoladek
Ramneek Kaur
RK
Johal
Samuel
S
Garcia-Nieto
Flavius
F
Pascut
Kevin M
KM
Shakesheff
Amir M
AM
Ghaemmaghami
Ioan
I
Notingher
D000199:Actins / Q000737:chemistry; D002478:Cells, Cultured; D016000:Cluster Analysis; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D054992:Immunological Synapses / Q000737:chemistry* / Q000648:ultrastructure; D007797:Laminin / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013601:T-Lymphocytes / Q000737:chemistry* / Q000648:ultrastructure
D000199:Actins; D007797:Laminin