Abstract
The acquired immune system, mainly composed of T and B lymphocytes, plays a key role in protecting the host from infection. It is important and technically challenging to identify cell types and their activation status in living and intact immune cells, without staining or killing the cells. Using Raman spectroscopy, we succeeded in discriminating between living T cells and B cells, and visualized the activation status of living T cells without labeling. Although the Raman spectra of T cells and B cells were similar, they could be distinguished by discriminant analysis of the principal components. Raman spectra of activated T cells with anti-CD3 and anti-CD28 antibodies largely differed compared to that of naïve T cells, enabling the prediction of T cell activation status at a single cell level. Our analysis revealed that the spectra of individual T cells gradually change from the pattern of naïve T cells to that of activated T cells during the first 24 h of activation, indicating that changes in Raman spectra reflect slow changes rather than rapid changes in cell state during activation. Our results indicate that the Raman spectrum enables the detection of dynamic changes in individual cell state scattered in a heterogeneous population.
Highlights
Fluorescence microscopy is an indispensable tool in biology and has greatly contributed to biological research for many years, as it is widely used for measuring cellular dynamics and status
CD4+ T cells recognize peptide-antigens presented on the major histocompatibility complex (MHC) with the T cell receptor (TCR) and produce cytokines to help or regulate immune cell activity, whereas B cells recognize soluble antigens with the B cell receptor and secrete specific antibodies against the antigen[10]
We have discriminated between T cells and B cells, and evaluated the extent of T cell activation by using Raman spectroscopy, which enabled the visualization of activated T cells without any labeling
Summary
To distinguish between T and B cells that showed similar spectra, we performed discriminant analysis of principal component (DAPC) on the obtained PCA scores. The difference in Raman spectra between the naïve and activated T cells can be further visualized by a PCA, where the naïve and activated T cells appear at different locations in the score plots (Fig. S2). Time course change in the logarithm of mean fluorescence intensity (MFI) of CD69 expression (Fig. 4B), which is an activation marker detected by flow cytometry using a specific antibody[16] This result indicates that changes in Raman spectra reflect the global changes in cell state upon cellular activation at the population level, and can quantitatively represent the activation process. Raman microscopy enables the discernment of T and B cells, and the monitoring of T cell activation in time; and space indicating that Raman microscopy has potential as an alternative method for fluorescent techniques such as flow cytometry
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