Non-contact mechanical and chemical analysis of single living cells by microspectroscopic techniques

Innovative label-free microspectroscopy, which can simultaneously collect Brillouin and Raman signals, is used to characterize the viscoelastic properties and chemical composition of living cells with sub-micrometric resolution. The unprecedented statistical accuracy of the data combined with the high-frequency resolution and the high contrast of the recently built experimental setup permits the study of single living cells immersed in their buffer solution by contactless measurements. The Brillouin signal is deconvoluted in the buffer and the cell components, thereby revealing the mechanical heterogeneity inside the cell. In particular, a 20% increase is observed in the elastic modulus passing from the plasmatic membrane to the nucleus as distinguished by comparison with the Raman spectroscopic marker. Brillouin line shape analysis is even more relevant for the comparison of cells under physiological and pathological conditions. Following oncogene expression, cells show an overall reduction in the elastic modulus (15%) and apparent viscosity (50%). In a proof-of-principle experiment, the ability of this spectroscopic technique to characterize subcellular compartments and distinguish cell status was successfully tested. The results strongly support the future application of this technique for fundamental issues in the biomedical field.

[1]  C. Kendall,et al.  Raman spectroscopy for medical diagnostics--From in-vitro biofluid assays to in-vivo cancer detection. , 2015, Advanced drug delivery reviews.

[2]  Satoshi Kawata,et al.  Label-free Raman observation of cytochrome c dynamics during apoptosis , 2011, Proceedings of the National Academy of Sciences.

[3]  Xiaohui Ni,et al.  Multicolor stimulated Raman scattering microscopy , 2012 .

[4]  A. Fontana,et al.  Elastic properties of permanently densified silica: A Raman, Brillouin light, and x-ray scattering study , 2010 .

[5]  D. Fioretto,et al.  Viscoelasticity of amyloid plaques in transgenic mouse brain studied by Brillouin microspectroscopy and correlative Raman analysis. , 2017, Journal of innovative optical health sciences.

[6]  A. Lenferink,et al.  Time Lapse Raman Imaging of Single Live Lymphocytes , 2011 .

[7]  Kristina Haase,et al.  Investigating cell mechanics with atomic force microscopy , 2015, Journal of The Royal Society Interface.

[8]  N Stone,et al.  Mechanical mapping with chemical specificity by confocal Brillouin and Raman microscopy. , 2014, The Analyst.

[9]  UK,et al.  High-Performance Versatile Setup for Simultaneous Brillouin-Raman Microspectroscopy , 2017, 1702.06707.

[10]  T. Woignier,et al.  Phonon attenuation in vitreous silica and silica porous systems , 2004 .

[11]  Jesse V Jokerst,et al.  A small animal Raman instrument for rapid, wide-area, spectroscopic imaging , 2013, Proceedings of the National Academy of Sciences.

[12]  J. R. Stevens,et al.  Raman and Brillouin scattering of LiClO4 complexed in poly(propylene‐glycol) , 1988 .

[13]  A. Fontana,et al.  Raman-scattering measurements of the vibrational density of states of a reactive mixture during polymerization: effect on the boson peak. , 2009, Physical review letters.

[14]  Daniele Zink,et al.  Nuclear structure in cancer cells , 2004, Nature Reviews Cancer.

[15]  Pilhan Kim,et al.  In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy. , 2011, Biophysical journal.

[16]  Liping Huang,et al.  Structure and Properties of Silica Glass Densified in Cold Compression and Hot Compression , 2015, Scientific Reports.

[17]  Marlan O Scully,et al.  Dual Raman-Brillouin Microscope for Chemical and Mechanical Characterization and Imaging. , 2015, Analytical chemistry.

[18]  H. You,et al.  Oncogenic Ras‐mediated downregulation of Clast1/LR8 is involved in Ras‐mediated neoplastic transformation and tumorigenesis in NIH3T3 cells , 2010, Cancer science.

[19]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[20]  Subra Suresh,et al.  Biomechanics and biophysics of cancer cells. , 2007, Acta biomaterialia.

[21]  Atsushi Miyawaki,et al.  Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression , 2008, Cell.

[22]  A. Modesti,et al.  Cellular Redox Imbalance and Changes of Protein S-glutathionylation Patterns Are Associated with Senescence Induced by Oncogenic H-Ras , 2012, PloS one.

[23]  J. Seely,et al.  Ultra-thin curved transmission crystals for high resolving power (up to E/ΔE = 6300) x-ray spectroscopy in the 6-13  keV energy range. , 2014, Optics letters.

[24]  Victoria J Allan,et al.  Light Microscopy Techniques for Live Cell Imaging , 2003, Science.

[25]  Peyman Milanfar,et al.  Pushing back the limits of Raman imaging by coupling super-resolution and chemometrics for aerosols characterization , 2015, Scientific Reports.

[26]  D. Docheva,et al.  Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy , 2007, Journal of cellular and molecular medicine.

[27]  T. Woignier,et al.  Acoustic attenuation in silica porous systems , 2003 .

[28]  Vladislav V. Yakovlev,et al.  Seeing cells in a new light: a renaissance of Brillouin spectroscopy , 2016 .

[29]  Byungkyu Kim,et al.  Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells , 2012, PloS one.

[30]  J. Rao,et al.  Nanomechanical analysis of cells from cancer patients. , 2007, Nature nanotechnology.

[31]  A. Giugni,et al.  Sound attenuation in a unexplored frequency region: Brillouin ultraviolet light scattering measurements in v-Si O 2 , 2005 .

[32]  Silvia Caponi,et al.  Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering , 2014, Journal of The Royal Society Interface.

[33]  Giuseppe Antonacci,et al.  Biomechanics of subcellular structures by non-invasive Brillouin microscopy , 2016, Scientific Reports.

[34]  X. Xie,et al.  Multicolored Stain-free Histopathology with Coherent Raman Imaging , 2012, Laboratory Investigation.

[35]  Glenn D. Boreman,et al.  Modulation Transfer Function in Optical and Electro-Optical Systems , 2001 .

[36]  Fabio Beltram,et al.  Simultaneous intracellular chloride and pH measurements using a GFP-based sensor , 2010, Nature Methods.

[37]  Zhaokai Meng,et al.  Subcellular measurements of mechanical and chemical properties using dual Raman‐Brillouin microspectroscopy , 2016, Journal of biophotonics.

[38]  Richard Superfine,et al.  Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. , 2011, Cancer research.

[39]  D. Fioretto,et al.  Progress in Liquid and Glass Physics by Brillouin Scattering Spectroscopy , 2012 .

[40]  William J. Polacheck,et al.  Noncontact three-dimensional mapping of intracellular hydro-mechanical properties by Brillouin microscopy , 2015, Nature Methods.

[41]  Kareem Elsayad,et al.  Mapping the subcellular mechanical properties of live cells in tissues with fluorescence emission–Brillouin imaging , 2016, Science Signaling.

[42]  L. Bernstein,et al.  Intraoperative brain cancer detection with Raman spectroscopy in humans , 2015, Science Translational Medicine.

[43]  G. Boreman Modulation Transfer Function , 1998 .

[44]  S. Pelli,et al.  Diagnostic techniques for photonic materials based on Raman and Brillouin spectroscopies , 2007 .

[45]  X. Xie,et al.  Video-Rate Molecular Imaging in Vivo with Stimulated Raman Scattering , 2010, Science.

[46]  Jan Toporski,et al.  Confocal Raman Microscopy , 2003, Microscopy and Microanalysis.

[47]  Carl Paterson,et al.  Spectral broadening in Brillouin imaging , 2013 .

[48]  Holly J. Butler,et al.  Using Raman spectroscopy to characterize biological materials , 2016, Nature Protocols.

[49]  Dan Fu,et al.  Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. , 2012, Journal of the American Chemical Society.

[50]  C. Musio,et al.  Raman micro-spectroscopy: a powerful tool for the monitoring of dynamic supramolecular changes in living cells. , 2013, Biophysical chemistry.

[51]  S. Yun,et al.  Confocal Brillouin microscopy for three-dimensional mechanical imaging. , 2007, Nature photonics.

[52]  Srinjan Basu,et al.  Label-free DNA imaging in vivo with stimulated Raman scattering microscopy , 2015, Proceedings of the National Academy of Sciences.

[53]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[54]  Rachel E. Factor,et al.  The nuclear envelope environment and its cancer connections , 2012, Nature Reviews Cancer.