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Non-label immune cell state prediction using Raman spectroscopy

View Article: PubMed Central - PubMed

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.

No MeSH data available.


(A) Pseudo-colored image of naïve (upper) and 48-h activated T cells. Images are pseudo-colored such that the naïve state is represented in blue whereas the activated state is in red. (B) Bright field image (left), Raman image (middle), and activation status (right) image of T cells activated for 24 h. The Raman image is color-coded as in Fig. 1, and the activation image is color-coded as in (A).
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f5: (A) Pseudo-colored image of naïve (upper) and 48-h activated T cells. Images are pseudo-colored such that the naïve state is represented in blue whereas the activated state is in red. (B) Bright field image (left), Raman image (middle), and activation status (right) image of T cells activated for 24 h. The Raman image is color-coded as in Fig. 1, and the activation image is color-coded as in (A).

Mentions: As each pixel constituting the overall Raman image contains its own Raman spectrum, the above analysis can be also applied to each of these spectra. The logarithm of probabilities that the spectra belong to naïve or activated cells, which are defined in the manner of quadratic discriminant analysis (QDA), were linearly converted to blue-green and red channels of 8-bit RGB color images (the blue and green channels were synchronized), so that a cell closer to the naïve/activated state was colorized in blue/red. Figure 5A shows a representative pseudo-colored image of naïve and activated T cells. The activation status of T cells was clearly visualized by this method, making it easy to correctly identify the activated T cells. To investigate whether activated T cells could be distinguished in heterogeneous populations using this method, CD4+ T cells that were activated for a short period were imaged with the Raman microscope. Figure 5B shows T cells activated for 12 h visualized in the same way as in Fig. 5A. Although the T cells in this view look similar in the bright-field image and in the Raman image (except for some apoptotic cells), activated T cells can be distinguished in the pseudo-colored image. Thus, Raman microscopy enables not only the discernment of T and B cells, but also 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.


Non-label immune cell state prediction using Raman spectroscopy
(A) Pseudo-colored image of naïve (upper) and 48-h activated T cells. Images are pseudo-colored such that the naïve state is represented in blue whereas the activated state is in red. (B) Bright field image (left), Raman image (middle), and activation status (right) image of T cells activated for 24 h. The Raman image is color-coded as in Fig. 1, and the activation image is color-coded as in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120326&req=5

f5: (A) Pseudo-colored image of naïve (upper) and 48-h activated T cells. Images are pseudo-colored such that the naïve state is represented in blue whereas the activated state is in red. (B) Bright field image (left), Raman image (middle), and activation status (right) image of T cells activated for 24 h. The Raman image is color-coded as in Fig. 1, and the activation image is color-coded as in (A).
Mentions: As each pixel constituting the overall Raman image contains its own Raman spectrum, the above analysis can be also applied to each of these spectra. The logarithm of probabilities that the spectra belong to naïve or activated cells, which are defined in the manner of quadratic discriminant analysis (QDA), were linearly converted to blue-green and red channels of 8-bit RGB color images (the blue and green channels were synchronized), so that a cell closer to the naïve/activated state was colorized in blue/red. Figure 5A shows a representative pseudo-colored image of naïve and activated T cells. The activation status of T cells was clearly visualized by this method, making it easy to correctly identify the activated T cells. To investigate whether activated T cells could be distinguished in heterogeneous populations using this method, CD4+ T cells that were activated for a short period were imaged with the Raman microscope. Figure 5B shows T cells activated for 12 h visualized in the same way as in Fig. 5A. Although the T cells in this view look similar in the bright-field image and in the Raman image (except for some apoptotic cells), activated T cells can be distinguished in the pseudo-colored image. Thus, Raman microscopy enables not only the discernment of T and B cells, but also 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.

View Article: PubMed Central - PubMed

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.

No MeSH data available.