Letter Cite This: J. Phys. Chem. Lett. 2017, 8, 5241-5245
pubs.acs.org/JPCL
Experimental Evaluation of the Density of Water in a Cell by Raman Microscopy Mizuki Takeuchi, Shinji Kajimoto, and Takakazu Nakabayashi* Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan S Supporting Information *
ABSTRACT: We report direct observation of a spatial distribution of water molecules inside of a living cell using Raman images of the O−H stretching band of water. The O− H Raman intensity of the nucleus was higher than that of the cytoplasm, indicating that the water density is higher in the nucleus than that in the cytoplasm. The shape of the O−H stretching band of the nucleus differed from that of the cytoplasm but was similar to that of the balanced salt solution surrounding cells, indicating less crowded environments in the nucleus. The concentration of biomolecules having C−H bonds was also estimated to be lower in the nucleus than that in the cytoplasm. These results indicate that the nucleus is less crowded with biomolecules than the cytoplasm.
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there are limited numbers of studies on water and hydrogenbonding networks of water inside of each organelle of living cells.9−14 In this study, we have succeeded in the direct evaluation of the amount of water molecules inside of a living cell using multi-confocal Raman microscopy, which leads to the quantitative evaluation of macromolecular crowding in a cell. By measuring Raman spectra of a cell around the O−H stretching band region and plotting its intensity as an image, we can visualize the spatial distribution of O−H bonds inside of a living cell and analyze the amount of water molecules in a cellular compartment. Hydrogen-bonding networks among water molecules or water and biological substances have also been examined by analyzing the shape and the peak position of the O−H stretching band. Here, we have compared the spatial distribution and molecular structures of water in the nucleus and in the cytoplasm of a single HeLa cell and a budding yeast cell. It has been shown that water density in the nucleus is higher than that in the cytoplasm in both cells. Raman measurements were performed using an inverted multi-confocal Raman microscope at an excitation wavelength of 532 nm (see the Supporting Information). The excitation light split into a square matrix array of 102 beamlets was focused onto a cultured cell on a glass-bottomed dish. Signals at all of the spots were transferred into an optical fiber bundle to be rearranged to a one-dimensional line. Then the single line of the Raman signals was introduced into a spectrograph, and 102 Raman spectra were detected at once without a scan of the excitation light. The advantage of the multi-confocal method is the execution of the fast acquisition of a Raman image to avoid
ntracellular environments are highly crowded with a large amount of biomolecules such as proteins and nucleic acids, which is called macromolecular crowding or molecular crowding.1−6 Evaluation of macromolecular crowding is important to understand biological activities in living cells. This is because the structures and functions of biomolecules are highly influenced by macromolecular crowding phenomena, such as the excluded volume effect, decrease in dielectric constant, and change in water hydration. Macromolecular crowding also strongly affects signal transduction by movement of biomolecules in cells.3−6 Fluorescence microscopy techniques measuring fluctuation or diffusion of exogenous fluorescent dyes are applicable to understand intracellular environments in terms of macromolecular crowding.5−8 However, these techniques are indirect methods that require dyes to detect cellular environments, and it is difficult to determine whether the change in the rate of fluctuation or diffusion of exogenous dyes is caused by the change in the macroscopic viscosity or by the change in transient binding of dyes with biomolecules. To understand macromolecular crowding and intracellular environments, it is necessary to directly observe molecules constituting intracellular environments. The quantitative evaluation of macromolecular crowding enables us to evaluate effects of macromolecular crowding on biological functions in a cell. Water is the most ubiquitous substrate in living cells that determines intracellular environments and governs biological reactions inside of a cell. Macroscopic intracellular parameters, such as viscosity and dielectric constant, and stabilization of biological molecules via water hydration depend on the number of water molecules per unit volume, that is, the density of water. Direct detection of water molecules inside of a cell is therefore important to understand intracellular environments. However, © XXXX American Chemical Society
Received: August 16, 2017 Accepted: October 4, 2017
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DOI: 10.1021/acs.jpclett.7b02154 J. Phys. Chem. Lett. 2017, 8, 5241−5245
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The Journal of Physical Chemistry Letters
Figure 1. Bright field (A) and corresponding Raman images (B,C) of a single HeLa cell. The Raman images were obtained by plotting the Raman intensity in the C−H stretching (B) and O−H stretching (C) band regions. (D) Raman spectrum of a HeLa cell.
prolonged laser irradiation inducing significant damage to the observed cell. Figure 1 shows a bright field image of a HeLa cell and the corresponding Raman images. The Raman images were constructed by plotting the integrated Raman intensity at 2846−2913 cm−1 (Figure 1B) and 3314−3673 cm−1 (Figure 1C). The areas are shown as bars in the Raman spectrum of a HeLa cell (Figure 1D). The Raman image in Figure 1B corresponds to a map of a lower-wavenumber region of the C− H stretching Raman band. In Figure 1C, a region corresponding to a higher-wavenumber region of the O−H stretching Raman band was plotted. It is clearly seen that these two Raman images show an opposite distribution of intensity from each other. In the C−H Raman image, the central part of the cell, corresponding to the nuclear region, showed lower intensity than that of the outer part of the cell (cytoplasm). In contrast, the O−H Raman image showed higher intensity in the nucleus than in the cytoplasm. This opposite behavior was observed irrespective of the z-axis when the interior of a cell was observed (Figure S1). The integrated Raman spectra of the nucleus and the cytoplasm of a HeLa cell are shown in Figure 2. The same area size was chosen in the two spectra to compare intensities of the Raman bands. The difference spectrum obtained by subtraction of the spectrum of the cytoplasm from that of the nucleus is also shown in the same figure. The quantitative analyses of both the intensity and the band shape are shown in the following discussion. The difference spectrum in the lower-wavenumber (fingerprint) region exhibits four negative peaks at 750, 1127, 1314, and 1583 cm−1 (Figure 2A). All of these peaks are attributed to cytochrome c, which shows enhanced Raman signals via resonance Raman effect as cytochrome c absorbs visible light around 532 nm (Figure S2).15,16 Cytochrome c is located in the inner membrane of mitochondrion in living cells. The negative sharp peaks in the difference spectrum and the lack of these peaks in the spectrum of the central part confirm that the central part contains much less cytochrome c than the outer part and is the nucleus of the cell. The outer part corresponds to the cytoplasm, which harbors mitochondria,
Figure 2. Difference Raman spectra (green) of a single HeLa cell obtained by subtracting the spectrum of the cytoplasm ((b) red) from the spectrum of the corresponding nucleus ((a) blue) in the fingerprint region (A) and in the high-wavenumber region (B).
resulting in resonance Raman signals from cytochrome c (Figure S3). The difference spectrum in the higher-wavenumber region (Figure 2B) shows a positive broad peak and a negative peak in the O−H stretching and the C−H stretching band region, respectively. Because the intensity of nonresonant Raman scattering is proportional to the number density of the observed compound, the present result indicates that the density of O− H bonds in the nucleus is higher than that of O−H bonds in the cytoplasm, while biomolecules having C−H bonds are less 5242
DOI: 10.1021/acs.jpclett.7b02154 J. Phys. Chem. Lett. 2017, 8, 5241−5245
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the spectrum of dried cells arises from all of the macromolecules inside of cells. The intensity of the O−H stretching band relative to that of the C−H stretching band is very small in the spectrum of dried cells, confirming that the O−H Raman intensity of a cell is derived mainly from water inside of a cell. Figure 3 clearly shows that the shape of the O−H stretching band in the nucleus differs from that in the cytoplasm, and the O−H band in the nucleus is similar to that of surrounding HBSS. This result indicates that hydrogen-bonding networks (molecular structures) of water in the nucleus are similar to that in the medium, which is consistent with the present result that the nucleus has a high density of O−H bonds and is less condensed with biomolecules. In the O−H band of the cytoplasm, the peak intensity at 3420 cm−1 seems to be relatively weaker than the other spectra in Figure 3. One possibility of the origin of this behavior is that the N−H stretching band of biomolecules in the cytoplasm cannot be neglected. The N−H band of biomolecules appears at around 3300 cm−1 (see the spectrum of dried cells), and the contribution of this band decreases in intensity at 3420 cm−1 relative to that at 3260 cm−1. This consideration is consistent with the result that the cytoplasm is more crowded with biomolecules than the nucleus. Furthermore, subtraction of the spectrum of the medium solution from that in the cytoplasm results in the appearance of a band that is similar to the N−H stretching band of dried cells (Figure S4). It may be further considered that the density of weakly hydrogen-bonded water in the cytoplasm is lower than that in the nucleus, which might be explained via enhancement of hydrogen bonds by hydrophobic solute molecules (hydrophobic hydration). In conclusion, analysis of the shape of the Raman band in the O− H stretching region indicates that the cytoplasm is more crowded with biomolecules, which induces the change in the shape of the Raman band in the O−H stretching region. We estimated the water densities in the nucleus and in the cytoplasm by integrating the bands in the O−H stretching region from 3400 to 3600 cm−1. Figure 4 shows the obtained integrated intensity divided by the integrated Raman intensity from the medium where cells were not observed as a reference. The size of the analyzed region was the same in the nucleus and in the cytoplasm. The contributions due to N−H and O−H bands of biomolecules were estimated by subtracting the
condensed in the nucleus. This implies that the nucleus of HeLa cells is sparser than their cytoplasm. Neither the negative nor positive bands in the difference spectrum (Figure 2B) coincide with the peak position and the bandwidth of corresponding Raman bands. In the C−H stretching band region, the negative peak in the difference spectrum is located at 2850−2950 cm−1, which is lower than the peak of the C−H stretching band. The C−H stretching Raman band can roughly be divided into a lower-wavenumber band less than 2950 cm−1, corresponding to saturated C−H bonds, and a higher-wavenumber band at around 3000 cm−1, corresponding to unsaturated C−H bonds. Furthermore, C−H bands of lipids are observed in the lower-wavenumber region, and those of proteins are in the higher-wavenumber region.17 The lower-wavenumber shift in the difference spectrum indicates that the concentration of saturated C−H bonds in the nucleus is lower than that in the cytoplasm, while that of unsaturated C−H bonds is almost the same between the nucleus and the cytoplasm. This implies that the observed difference in the C−H stretching region is mainly due to a lower concentration of lipid molecules in the nucleus. In the O−H stretching band region, the positive peak in the difference spectrum is located at around 3460 cm−1. The O−H stretching Raman band of pure water exhibits a broad peak at 3420 cm−1 with a shoulder at around 3230 cm−1 and can be divided into higher-wavenumber components, assigned to weakly hydrogen-bonded water, and lower-wavenumber components, assigned to strongly hydrogen-bonded water.18 Therefore, the positive peak at 3460 cm−1 in the difference spectrum is due to the difference in hydrogen bonds of water and/or in the Raman bands of biomolecules between the nucleus and the cytoplasm. Figure 3 quantitatively compares the shapes of the average Raman spectra of the nucleus and cytoplasm of HeLa cells with
Figure 3. Average Raman spectra of HeLa cells in the nucleus (blue) and in the cytoplasm (red), together with those of the HBSS medium where cells were not observed (green) and dried HeLa cells (black). The background of each spectrum was fitted with a polynomial function and was subtracted as a baseline.
that of Hanks’s balanced salt solution (HBSS) medium measured at the region where cells were not observed in the dishes. These spectra were normalized with respect to the intensity of the O−H band at ∼3260 cm−1. The Raman spectrum of dried HeLa cells is also shown in the same figure, representing the intensities of C−H, N−H, and O−H stretching bands of macromolecules in cells. It is noted that
Figure 4. Integrated O−H stretching Raman intensities of the cytoplasm and the nucleus of HeLa cells divided by the integrated O− H stretching Raman intensity of the cell medium. The intensities due to N−H and O−H bands of biomolecules were subtracted in the analysis. 5243
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of the crucial parameters in understanding intracellular circumstances and biological application such as distinguishing of cancer cells from normal cells and quality control in a cell culture. In conclusion, we have investigated the density and hydrogen-bonding networks of water in each cellular compartment using spectra and images of the O−H stretching Raman band. The images clearly indicate that the water density in the nucleus is higher than that in the cytoplasm. The environment is less crowded and hydrophobic hydration, that is, the distortion of hydrogen-bonding networks due to the presence of hydrophobic molecules, is less dominant in the nucleus as compared with that in the cytoplasm. The shape of the O−H stretching band is similar between the nucleus and the medium, supporting the above conclusion. Recently, O−H stretching Raman bands of cells were used to evaluate the ionic transport between intracellular and extracellular fluids.14 We believe that Raman imaging in the O−H stretching band region could be a new powerful method for in vivo and label-free analysis of intracellular environments.26
spectrum of the medium solution from those of cells (Figure S4). Then the evaluated contributions were removed in the analysis of the Raman intensities in Figure 4. The relative intensity of the nucleus is near unity, suggesting that the observed water density in the nucleus is similar to that in the medium. The relative intensity of the cytoplasm is estimated to be 3% smaller than that of the nucleus. This result concludes that the water density in the cytoplasm is ∼3% smaller than that in the nucleus. The Raman images of budding yeast cells were also observed in the C−H and O−H stretching regions (Figure S5). The obtained difference spectrum is similar to that of a HeLa cell, and a positive and a negative peak are observed in the O−H stretching and the C−H stretching band region, respectively. This result indicates that the nucleus of a yeast cell is also less crowded than the cytoplasm, and the water density is higher in the nucleus than that in the cytoplasm. In the C−H stretching region, the negative peak is relatively larger than that observed in HeLa cells and is located at ∼2935 cm−1, which is almost the same as that of the corresponding Raman bands of the nucleus and the cytoplasm. This implies that the difference in the concentration of biomolecules between the nucleus and the cytoplasm is slightly larger in yeast cells than that in HeLa cells. In the O−H stretching region, on the other hand, the peak position of the difference band is similar to that observed in HeLa cells, suggesting that the difference in the hydrogenbonding network between the nucleus and the cytoplasm is similar between yeast cells and HeLa cells. We have shown here that the water density is higher in the nucleus than that in the cytoplasm, and the magnitude of the difference is estimated to be 3%. This means that the concentration of biomolecules in the nucleus is lower than that in the cytoplasm. The viscosity and the dielectric constant of an aqueous solution of macromolecules have a tendency to increase and decrease, respectively, with increasing concentration of added macromolecules. The present results therefore indicate that the macroscopic viscosity in the nucleus is lower than that in the cytoplasm and the dielectric constant of the environment is higher in the nucleus than that in the cytoplasm. These findings seem to be difficult to be obtained by other methods such as fluorescence and optical tomography, and we believe that the present result will become the basis of the evaluation of dynamics and molecular structures of biomolecules in cellular compartments in terms of macromolecular crowding.1−8 The present result is consistent with the recent measurement of the refractive index or fluorescence dynamics of a dye in a cell.19−23 The refractive index was shown to be smaller in the nucleus than that in the cytoplasm, suggesting a low concentration of organic molecules in the nucleus.19,20 On the basis of the position of the emission maximum of an exogenous dye, the dielectric constant of the nucleus was suggested to be larger than that of the cytoplasm.21−23 The viscosities of the nucleus and cytoplasm were also estimated to be ∼13 and ∼14.5 cP, respectively.22 It is noted that the present value is the average of the intracellular environment, which slightly fluctuates in seconds.24 The present study directly evaluates the amount of water molecules in cells and conclusively shows that the nucleus is less crowded than the cytoplasm. The water density in each cellular compartment should reflect the intracellular viscosity, polarity, and other macroscopic properties at each compartment related to macromolecular crowding.25 The new parameter “water density in a cell” and its spatial distribution may become therefore one
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.7b02154. Materials and methods details and supplementary results, including Raman images at different depths, absorption spectra, and difference Raman spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Takakazu Nakabayashi: 0000-0003-2942-2189 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are very grateful to President Katsuo Morita, Mr. Hiroyuki Watabe, and Dr. Hideaki Takechi at Tokyo Instruments Inc. for their great support on the multi-confocal Raman system. This work was partly supported by a Grant-inAid for Scientific Research on Innovative Area (No. 4602) from the Ministry of Education, Culture, Sports, Science and Technology in Japan and JSPS KAKENHI Grant Number JP15H03518.
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