Raman and infrared microspectral imaging of mitotic cells

Abstract

We report the first ever Raman and infrared microspectroscopic images of human cells at different stages of mitosis. These spectroscopic methods monitor the distribution of condensed nuclear chromatin, and other biochemical components, utilizing inherent protein and DNA spectral markers, and, therefore, do not require the use of any stains. In conjunction with previously reported data from the G1, S, and G2 phases of the cell cycle, the complete cell division cycle has now been mapped by spectroscopic methods. Although the results reported here do not offer new insights into the distribution of biochemical components during mitosis, the recognition of cell division without the use of stains, and the possibility of doing so on living cells, may be useful for an automatic, spectroscopic determination of the proliferation rates of cells and tissues. Spectral images were constructed by plotting spectral intensities of DNA or protein versus the coordinates from which spectra were recorded. We found that both Raman and infrared intensities depend on the overall chromatin density variation among the individual subphases of mitosis.

Fig. 1

(A) Visual photomicrograph of unstained HeLa cell, grown on a CaF₂ substrate. (B) Infrared spectral image of the cell shown in (A), based on protein absorption intensity. The spatial resolution is about 12 μm at 1000 cm⁻¹ absorption. Light gray hues indicate low intensity, while black indicates highest intensity. (C) Hierarchical Cluster Map of the data set shown in (B). (D) Infrared spectrum of the nuclear (black) region. (E) Dendrogram for the clustering process used to produce (C). The dissimilarity between spectra increases along the Y axis.

Fig. 2

(A) Visual photomicrograph of an unstained HeLa cell, grown on a CaF₂ substrate. (B) Raman spectral image of the region indicated by the black square, based on the Raman scattered intensity at 785 cm⁻¹. Black indicates low intensity, to red, to white (highest intensity). (C) Pseudo-color map of same cell, based on hierarchical cluster analysis. (D, E, F) Single point Raman spectra from within the cytoplasm, the nucleus, and the nucleoli, respectively.

Fig. 3

Photomicrographs and Raman and fluorescence (DAPI stain) images of HeLa cells during various phases of mitosis. (Top row) prophase, (second row) metaphase, (third row) anaphase, and (bottom row) late telophase. All images are collected at 40× magnification. (Column 1) Phase-contrast images of live cells in culture. (Column 2) Raman scattering intensity plots for the DNA scattering intensities. The colors range from black (low intensity), to red, to white (high intensity). (Column 3) Raman scattering intensity plots for the protein scattering intensities. The colors range from black (low intensity), to green, to yellow (high intensity). (Column 4) Fluorescence images of the DAPI-stained cells.

Fig. 4

Raman spectra of (A) cytoplasm and (B) DNA/chromatin complex during the anaphase of mitosis. Major bands due to DNA are indicated by their wavenumbers.

Fig. 5

Phase-contrast images of HeLa cells in (A) metaphase and (C) anaphase. (B) Infrared spectral map based on the amide I absorption intensities, of the cell shown in (A). In the center of the nucleus, the largest intensity correlates with the position of the chromatin. Dark blue indicates low intensity, and yellow to red indicate higher and highest intensity, respectively. (D) Resolution-enhanced infrared spectral map based on the 2925 cm⁻¹ absorption region of the cell shown in (C). The two chromatin regions of the cell in anaphase are detectable. The color scheme is as in (B).