Extra-Cellular Water States Probed by

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A: New Tools and Methods in Experiment and Theory

Characterization of Intra/Extra-Cellular Water States Probed by Ultrabroadband Multiplex Coherent AntiStokes Raman Scattering (CARS) Spectroscopic Imaging Mutsuo Nuriya, Hiroaki Yoneyama, Kyosuke Takahashi, Philippe Leproux, Vincent Couderc, Masato Yasui, and Hideaki Kano J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b03018 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 8, 2019

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The Journal of Physical Chemistry

Characterization of Intra/Extra-Cellular Water States Probed by Ultrabroadband Multiplex Coherent AntiStokes Raman Scattering (CARS) Spectroscopic Imaging Mutsuo Nuriya 1, 2, 3, 4, #, *, Hiroaki Yoneyama 5, Kyosuke Takahashi 5, Philippe Leproux 6 ,7, Vincent Couderc 6, Masato Yasui 1, 2 & Hideaki Kano 2, 5 ,8 ,9 #, *.

1Department

of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku,

Tokyo 160-8582, Japan 2Keio

Advanced Research Center for Water Biology and Medicine, Keio University, 2-15-45,

Mita, Minato, Tokyo, 108-8345, Japan 3Precursory

Research for Embryonic Science and Technology (PRESTO), Japan Science and

Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan 4Graduate

School of Environment and Information Sciences, Yokohama National University, 79-

1 Tokiwadai, Hodogaya, Yokohama, Kanagawa 240-8501, Japan

ACS Paragon Plus Environment

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5Department

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of Applied Physics, Graduate School of Pure and Applied Sciences, University of

Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan 6Institut

de Recherche XLIM, UMR CNRS No. 7252, 123 Avenue Albert Thomas, 87060

Limoges CEDEX, France 7LEUKOS,

8Institute

37 Rue Henri Giffard, 87280 Limoges, France

of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-

8573, Japan 9Tsukuba

Research Center for Energy Materials Science (TREMS), University of Tsukuba, 1-1-1

Tennodai, Tsukuba, Ibaraki 305-8571, Japan

# These authors contributed equally to this work.

* Corresponding Authors [email protected] (MN), [email protected] (HK)


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ABSTRACT

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ~10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside cells, suggesting the existence of stronger hydrogen bonds inside cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside cells and thereby induces dehydration of cells. Under this treatment, the red shift of O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.

KEYWORDS CARS, water, cell, O-H stretching, Raman, microscopy


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INTRODUCTION Water constitutes nearly 60% of the total body weight of humans and other mammals, and thus represents the most abundant molecule in living organisms. As such, water molecules interact with most of the intracellular and extracellular biomolecules, thereby affecting their bioactivities 1.

Indeed, even slight changes in the body water content can have severe physiological

consequences in humans. Despite this importance, in-depth imaging analysis of the chemical state of water molecules in living cells has been challenging because their small size is not conducive for detection by conventional labeling techniques. In this regard, label-free Raman microscopy could hold great promise in the visualization of water. Raman microscopy can be utilized to visualize small bioactive molecules whose distributions and dynamics would be severely affected by the addition of fluorescent dyes, and water is arguably one of the most important bioactive molecules of this sort 2-4. However, the low efficiency of conventional spontaneous Raman scattering limits its applications in biological research. In particular, the long exposure time and/or high-power laser illuminations result in slow imaging and serious phototoxicity to living cells. These limitations have been dramatically overcome by the use of coherent Raman scattering instead of spontaneous Raman 5-8, which improved the signal intensity by a factor of at least 100 9. Arguably, the best of the available techniques for this application is coherent anti-Stokes Raman scattering (CARS) imaging, which was first applied to biological research in the 1980s 10 and 1990s 11, 12. Despite its great potential, the main drawback of CARS is that the signal is only generated on the functional group that exactly matches the energy difference between the pump and Stokes lasers, and thus it can only detect and visualize one specific chemical bond at a time. Although stepwise changes in the laser wavelength and subsequent imaging are feasible, the


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limited spectral coverage remains a barrier to wider applications 13, 14. However, the development of supercontinuum lasers that contain photons covering a wide wavelength spectrum in ultrashort pulses could help to overcome this issue 15-17. Using this laser as a Stokes beam, multiplex CARS can probe a wide range of chemical information simultaneously, thereby dramatically improving both the temporal resolution and spectral coverage. Therefore, multiplex CARS spectroscopic imaging can serve as an unprecedented powerful tool for H2O chemical imaging of living cells; however, this potential of CARS has not yet been realized. In this study, we applied our home-built ultrabroadband multiplex CARS microscopic system with a supercontinuum light source 18, 19 to visualize the molecular distribution inside living cells at high spatiotemporal and spectral resolutions.


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EXPERIMENTAL METHODS CHO cells Chinese hamster ovary (CHO) cells originally obtained from American Type Culture Collection (ATCC) were maintained as reported previously 20, 21. For imaging experiments, CHO cells were plated on 35 mm glass-bottom dishes coated with 100 g/mL poly-L-lysine in borate buffer (0.1M, pH 8.5) at a density of 2 – 5 × 104 cells / dish. Imaging was performed 1 – 3 days after plating.

CARS imaging The multiplex coherent anti-Stokes Raman scattering system developed by our group has been reported elsewhere 18, 22. A Q-switched microchip Nd:YAG laser was used as a master laser source. Part of the output of the master laser was used for ω1 laser (pump laser). The other was introduced into a photonic crystal fiber (PCF) to generate supercontinuum (SC) radiation, which was used as ω2 laser. Both the ω1 and ω2 laser beams were superimposed by a notch filter and then introduced into a modified inverted microscope (Nikon: ECLIPSE Ti). The sample was placed upon a piezoelectric stage (PZT, Mad City Lab: Nano-LP200) for position selection. The laser beams are tightly focused using a water-immersion objective lens (CFI Plan Apo 60x NA 1.27 (Nikon)). The CARS signal was collected by the second objective lens and detected by a spectrometer (Princeton Instruments: LS785) and CCD camera (Princeton Instruments: PIXIS 100BR). The exposure time was 50 ms. For data analysis, the maximum entropy method (MEM) was used to retrieve the imaginary part of (3) from the raw multiplex CARS spectra 23, 24. MEM


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does not require any a priori information on the vibrational resonance structure of the sample to extract quantitative spectral information from multiplex CARS spectra in congested spectral regions. Details of this method are described in the literature 23, 24 . Imaging was performed at room temperature (~20 °C) in the imaging buffer containing 125 mM NaCl, 5 mM KCl, 10 mM dextrose, 10 mM HEPES, 1 mM MgCl2, and 2 mM CaCl2, pH 7.3. Osmolality of this imaging buffer was ~ 300 mOsm.

Pharmacology 1M D(-)-mannitol (Wako Pure Chemical) in imaging buffer was prepared with ~1,300 mOsm. To induce osmolality change, 900 L of 1M mannitol in imaging buffer was gently added to the dish under observation containing 2,000 L of imaging buffer. This procedure changed the osmolality of the extracellular solution from ~300 mOsm to ~600 mOsm.

Data analyses Data were analyzed using Igor (WaveMetrics) and MATLAB (Mathworks) by custom-written software. Briefly, the program reads the original 3D data (x, y, and Raman shift) and calculates mean intensities within the selected regions of interest (ROI). In the case of semi-automatic separation of intracellular and extracellular regions, signals corresponding to the C-H stretching region were divided by those of O-H for normalization among different positions in the field of views, which was then used to separate intracellular and extracellular regions by the threshold


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value and calculate mean Raman spectra of intracellular and extracellular regions. In the case of comparison between the shapes of spectra, the data obtained from different ROIs were normalized by adjusting the values by dividing them by the differences between maximum and minimum values in the Raman shift region from 3,200 cm-1 to ~4,000 cm-1. In other cases, the original values were used for calculations. For statistical analyses, Wilcoxon signed rank tests were used for comparison between groups (OriginPro).


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RESULTS AND DISCUSSION Chinese hamster ovary (CHO) cells grown on glass-bottom dishes 20, 21 were placed onto the microscope system, irradiated by the temporally synchronized pump and broadband Stokes pulses, and the resulting multiplex CARS signals were collected by a spectrometer (Figure 1A). To probe the chemical state of water in living cells, we optimized the system so that it can record the Raman shift in the range of 500–4,000 cm-1, covering the fingerprint regions to the whole spectrum of the O-H stretching bond (Figure 1B). Scanning was performed by moving the piezo stage, normally at 50 msec per point. In this protocol, 101 × 101-pixel images covering the whole spectrum can be obtained within ~8 min. Figure 1C shows a representative image of CHO cells at different wavenumbers, whose related assignments have been previously described 25. These images clearly demonstrate that this system can effectively perform wide-spectrum chemical imaging of living mammalian cells at high spatiotemporal and spectral resolutions.


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To gain insights into the chemical properties of water molecules inside and outside living cells, we next focused our attention to the spectra corresponding to the O-H stretching mode. Comparison of the spectra inside and outside cells was performed using two methodologies. First, the intracellular and extracellular regions were semi-automatically separated according to the relative amplitude of the C-H stretching signal (2,930–2,940 cm-1) over the O-H stretching signal


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(3,420–3,430 cm-1), where higher values correspond to more C-H-rich intracellular regions (Figure 2A). This semi-automatic separation algorithm separates intracellular and extracellular regions that nicely match the visual inspections of the original image. Moreover, this method is objective and can analyze the overall differences between the two regions. In the second method, the regions of interest (ROIs) were selected based on the C-H signals (Figure 2B). This method allows for analyses of particular regions of interest such as the nucleus and particular subregions in the cytosol, albeit it is more susceptible to variations between the selected region and the signal-to-noise ratio is reduced when smaller regions are selected. Regardless of their differences, these two methodologies showed the same tendencies when the average intensities of intracellular and extracellular regions were compared (Figure 2). The successful separations of these two regions were confirmed by a clear signal of the C-H stretching mode in the intracellular spectra that was virtually absent in the spectra corresponding to the extracellular region. The spectra were compared in two ways: with the original CARS signal amplitude that reflects the relative density of the compounds, and using a normalization procedure to more effectively compare the shapes of spectra (Figure 2A, 2B).


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Comparison of spectra obtained from intracellular and extracellular regions revealed consistent differences in their shapes with a tendency of a red-shift of the spectra and broadening of the shoulder at lower wavenumber regions (3,200–3,400 cm-1). To quantitatively analyze these differences, we first analyzed the signal of the O-H stretching mode in detail; the whole spectrum that apparently has two shoulders was decomposed into two Gaussian signals. Indeed, the O-H stretching mode was well fit by the sum of the two Gaussian shapes with peaks at 3,209 cm-1 (peak 1) and 3,416 cm-1 (peak 2). Based on this analysis, we next quantified the ratio of these two peaks (peak 1/peak 2). Although the absolute values varied, likely reflecting both biological and instrumental variations, comparison of data from inside and outside cells revealed consistent robust differences between the peak ratios (Figure 2C) (p = 0.00195, n = 10, Wilcoxon signed rank test). These results demonstrate that water molecules inside cells are relatively more redshifted, suggesting that the O-H stretching is weaker inside cells compared to outside owing to a stronger hydrogen bond. Finally, we sought to manipulate the state of water inside/outside cells for the context of medical applications. One of the best characterized pathophysiological conditions involving disturbance of water homeostasis is the state of edema, involving the abnormal accumulation of water in tissues. Mannitol is widely used for the treatment of brain edema 26. Mannitol is a saccharide that is innate to cells, and, importantly, does not permeate the plasma membrane and thus remains outside cells when applied to the extracellular solution. As a result, the osmolality outside cells becomes higher than that inside, which induces the net efflux of water molecules from cells. Therefore, to gain insights into the pharmacological action of mannitol in the context of water, we characterized the effect of mannitol treatment on the water state inside/outside living cells.


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When mannitol was introduced to the extracellular solution to increase the osmolality from ~300 mOsm to ~600 mOsm, shrinkage of cells was observed, confirming the dehydrating effect of mannitol on cells (Figure 3A). Outside the cells, the C-H stretching signal that was undetectable in the original extracellular solution appeared after introduction of mannitol, as predicted from the fact that mannitol contains C-H bonds (Figure 3B). Alongside these predicted changes, a prominent change in the spectrum was observed inside the cell in which the relative amplitude of the O-H signal compared to the C-H signal dropped dramatically after mannitol treatment (Figure 3B). As C-H species inside cells does not change after mannitol addition, this means that the O-H density decreased after mannitol treatment. Although many intracellular molecules, including nucleic acids, saccharides, and amino acids, contain O-H bonds, these molecules are not readily capable of moving from inside to outside cells. Rather, water is the predominant O-H bond-carrying molecule that moves upon mannitol exposure. Therefore, the prominent reduction of the O-H signal after mannitol treatment suggests that the origin of the O-H signal under normal conditions is mainly the intracellular water molecules rather than the hydroxyl groups of other intracellular molecules, consistent with a recent report based on spontaneous Raman microscopy analysis 27. Moreover, detailed inspection revealed that the shape of the O-H signal change to show a broader peak owing to the higher shoulder at lower wavenumber regions (3,200–3,400 cm-1) after mannitol treatment (Figure 3B, 3C). Indeed, in a limited number of cases when spectra were successfully obtained from the same field of views, the peak ratio values increased upon mannitol treatment (Figure 3C, p = 0.125, n = 5, Wilcoxon signed rank test). This difference matches well with the differences in O-H spectra observed between intracellular and extracellular regions (Figure 2B, 2C), further supporting the idea that these changes reflect


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stronger hydrogen bonds; that is, dehydration of water by mannitol makes the intracellular environment more crowded, which then increases the strength of hydrogen bonds of water molecules. Furthermore, because the effects of mannitol are mainly limited to water molecules, the above observations add strong experimental evidence to support that the differences between intra- and extra-cellular spectra and changes induced by mannitol reflect those of water molecules but not others containing O-H and/or N-H groups 27, 28.


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In conclusion, we have demonstrated the chemical imaging of intracellular and extracellular water at high spatiotemporal and spectral resolution using ultrabroadband multiplex CARS imaging, which revealed differences in the chemical states of water inside and outside cells


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under normal condition and upon mannitol treatment. Whereas detailed characterization of the molecular basis of these changes is beyond the scope of this study, the ability to measure the spectral profiles of water inside and outside cells under various conditions should allow our new system to contribute to the fields of physical chemistry and biology of water molecules 1-4, 29-31. In fact, although detailed knowledge on the chemical state of water in living organisms is lacking, owing to its predominance in the body, water-based MRI technology is currently widely used for diagnostic purposes, most notably for cancer diagnosis. In spite of its general uses, theoretical understanding behind the ability of this technique to discriminate between healthy and tumor tissues remains poor. This knowledge gap is largely attributed to the lack of detailed spectral information and sufficient spatiotemporal resolutions. Accordingly, our CARS-based analysis shows great promise toward improving the knowledge gap of the chemical state of water in living organisms with detailed spectral information at sufficient spatiotemporal resolutions. Importantly, as a multiphoton imaging technique using near-infrared lasers, our system should allow for deep-tissue high-spatiotemporal-resolution CARS imaging, which should be of great benefit in applying this system for tissue imaging. Furthermore, backscattering of CARS signals have been successfully detected from live animals 32. Therefore, we expect that our system can be further applicable for in vivo measurements in the future, providing a major contribution to basic and clinical research of various diseases, including cancer.


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CONCLUSIONS In this study, we have established a bio-compatible ultrabroadband CARS microscopy system tuned to analyze the whole O-H stretching mode that revealed the spectral differences between intra- and extra-cellular water molecules in living mammalian cells. Furthermore, the advantage of this system was exploited to investigate the effects of mannitol addition to the extracellular solution that mimics the pharmacological interventions against edema. Therefore, the methodology reported here is applicable to a wide range of biological studies and should serve as a powerful tool to improve our understanding of water molecules in the context of biology and medicine.


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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI (26282117, 18H02000, 18K18444 to H.K. and 16K07065 & ResonanceBio 16H01434 to M.N.) and JST PRESTO (JPMJPR17G6 to M.N.). The authors gratefully acknowledge J. Ukon, Ukon Craft Science, Ltd., for assisting with a fruitful collaboration between Japanese and French laboratories, T. Yamada for her help in maintaining the CHO cells, Y. Koyama for his help in writing the MATLAB analysis program. Finally, the authors greatly acknowledge D. Kojic for helpful discussions and careful reading of the manuscript.


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Extra-Cellular Water States Probed by

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