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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.

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Raman spectrum from T cells and B cells.(A) Fingerprint region of the averaged Raman spectra obtained from 96 naïve T cells (blue) and 60 B cells (green). (B) The first 7 loading vectors calculated by PCA. (C,D) Result of DAPC of Raman spectra of naïve T and B cells. (C) Scatter plot of LDA scores for naïve T cells (blue) and B cells (green) along (D) the first discriminant axis (F1 vector). Each dot in (C) represents a single cell.
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f2: Raman spectrum from T cells and B cells.(A) Fingerprint region of the averaged Raman spectra obtained from 96 naïve T cells (blue) and 60 B cells (green). (B) The first 7 loading vectors calculated by PCA. (C,D) Result of DAPC of Raman spectra of naïve T and B cells. (C) Scatter plot of LDA scores for naïve T cells (blue) and B cells (green) along (D) the first discriminant axis (F1 vector). Each dot in (C) represents a single cell.

Mentions: First, we compared the Raman spectra of T and B cells. Raman spectra were obtained using a home-built slit scanning microscope based on an inverted microscope (Ti Series, Nikon), equipped with a spectrometer (MK-300, Bunkou Keiki), as previously reported3. Figure 1A and C show Raman images of T and B cells, respectively, and Fig. 1B,D show representative Raman spectra from the cytosol and the nucleus. Although the Raman signal is stronger in the cytosolic region, in this study we averaged the spectra from the whole-cell region, and treated it as the Raman spectrum from a single cell. To compare the Raman spectra of T and B cells, Raman spectra from 96 T cells and 60 B cells from a DO11.10 mouse were averaged within cell-type, and were compared (Fig. 2A). The Raman spectra of these two cell-types were similar; however, differences were observed in some peaks, such as those at 1089, 1332, 1375, and 1483 cm−1.


Non-label immune cell state prediction using Raman spectroscopy
Raman spectrum from T cells and B cells.(A) Fingerprint region of the averaged Raman spectra obtained from 96 naïve T cells (blue) and 60 B cells (green). (B) The first 7 loading vectors calculated by PCA. (C,D) Result of DAPC of Raman spectra of naïve T and B cells. (C) Scatter plot of LDA scores for naïve T cells (blue) and B cells (green) along (D) the first discriminant axis (F1 vector). Each dot in (C) represents a single cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Raman spectrum from T cells and B cells.(A) Fingerprint region of the averaged Raman spectra obtained from 96 naïve T cells (blue) and 60 B cells (green). (B) The first 7 loading vectors calculated by PCA. (C,D) Result of DAPC of Raman spectra of naïve T and B cells. (C) Scatter plot of LDA scores for naïve T cells (blue) and B cells (green) along (D) the first discriminant axis (F1 vector). Each dot in (C) represents a single cell.
Mentions: First, we compared the Raman spectra of T and B cells. Raman spectra were obtained using a home-built slit scanning microscope based on an inverted microscope (Ti Series, Nikon), equipped with a spectrometer (MK-300, Bunkou Keiki), as previously reported3. Figure 1A and C show Raman images of T and B cells, respectively, and Fig. 1B,D show representative Raman spectra from the cytosol and the nucleus. Although the Raman signal is stronger in the cytosolic region, in this study we averaged the spectra from the whole-cell region, and treated it as the Raman spectrum from a single cell. To compare the Raman spectra of T and B cells, Raman spectra from 96 T cells and 60 B cells from a DO11.10 mouse were averaged within cell-type, and were compared (Fig. 2A). The Raman spectra of these two cell-types were similar; however, differences were observed in some peaks, such as those at 1089, 1332, 1375, and 1483 cm−1.

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.


Related in: MedlinePlus