Non‐invasive time‐course imaging of apoptotic cells by confocal Raman micro‐spectroscopy

Confocal Raman micro-spectroscopy (CRMS) was used to measure time-course spectral images of live cells undergoing apoptosis without using molecular labels or other invasive procedures. Human breast cancer cells (MDA-MB-231) were exposed to 300 µM etoposide to induce apoptosis, and Raman spectral images were acquired from the same cells at 2-h intervals over a period of 6 h. The purpose-built inverted confocal Raman micro-spectrometer integrated an environmental enclosure and wide-field fluorescence imaging. These key instrumental elements allowed the cells to be maintained under sterile physiological conditions (37 °C, 5% CO2) and enabled viability and apoptosis assays to be carried out on the cells at the end of CRMS measurements. The time-course spectral images corresponding to DNA Raman bands indicated an increase in signal intensity in apoptotic cells, which was attributed to DNA condensation. The Raman spectral images of lipids indicated a high accumulation of membrane phospholipids and highly unsaturated non-membrane lipids in apoptotic cells. This study demonstrates the potential of CRMS for label-free time-course imaging of individual live cells. This technique may become a useful tool for in vitro toxicological studies and testing of new pharmaceuticals, as well as other time-dependent cellular processes, such as cell differentiation, cell cycle and cell–cell interactions. Copyright © 2010 John Wiley & Sons, Ltd.

[1]  Ronald A. Li,et al.  Label-free separation of human embryonic stem cells and their cardiac derivatives using Raman spectroscopy. , 2008, Analytical chemistry.

[2]  K Bergman,et al.  Characterization of photodamage to Escherichia coli in optical traps. , 1999, Biophysical journal.

[3]  H. Takeuchi,et al.  Lipid structure of cytotoxic granules in living human killer T lymphocytes studied by Raman microspectroscopy. , 1997, Biochimica et biophysica acta.

[4]  M. Berns,et al.  Wavelength dependence of cell cloning efficiency after optical trapping. , 1996, Biophysical journal.

[5]  D. Green,et al.  Measuring apoptosis at the single cell level. , 2008, Methods.

[6]  Satoshi Kawata,et al.  Raman microscopy for dynamic molecular imaging of living cells. , 2008, Journal of biomedical optics.

[7]  Y. Hannun,et al.  Inhibition of Caspases Inhibits the Release of Apoptotic Bodies: Bcl-2 Inhibits the Initiation of Formation of Apoptotic Bodies in Chemotherapeutic Agent-induced Apoptosis , 1999, The Journal of cell biology.

[8]  Abdelilah Beljebbar,et al.  Raman spectral imaging of single living cancer cells: a preliminary study. , 2009, The Analyst.

[9]  Menghong Sun,et al.  Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells. , 2006, Optics letters.

[10]  I Barba,et al.  The relationship between nuclear magnetic resonance-visible lipids, lipid droplets, and cell proliferation in cultured C6 cells. , 1999, Cancer research.

[11]  Gavin Jell,et al.  Non‐invasive analysis of cell cycle dynamics in single living cells with Raman micro‐spectroscopy , 2008, Journal of cellular biochemistry.

[12]  James P Freyer,et al.  Raman spectroscopy detects biochemical changes due to proliferation in mammalian cell cultures. , 2005, Biophysical journal.

[13]  Hiro-o Hamaguchi,et al.  Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy. , 2005, Biochemistry.

[14]  Molly M Stevens,et al.  Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation. , 2008, Biophysical journal.

[15]  C. Otto,et al.  The intensity of the 1602 cm-1 band in human cells is related to mitochondrial activity , 2009 .

[16]  P. Bozza,et al.  Leukocyte lipid body formation and eicosanoid generation: cyclooxygenase-independent inhibition by aspirin. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Sledge.  Etoposide in the management of metastatic breast cancer , 1991, Cancer.

[18]  M. Diem,et al.  Spectroscopy , 2007, Acta Neuropsychiatrica.

[19]  R. Coleman,et al.  Enzymes of triacylglycerol synthesis and their regulation. , 2004, Progress in lipid research.

[20]  Guiwen Wang,et al.  Raman spectroscopic analysis of apoptosis of single human gastric cancer cells , 2009 .

[21]  L L Hench,et al.  Bioactive glass-induced osteoblast differentiation: a noninvasive spectroscopic study. , 2008, Journal of biomedical materials research. Part A.

[22]  L L Hench,et al.  Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy , 2004, Journal of The Royal Society Interface.

[23]  Max Diem,et al.  Shedding new light on the molecular architecture of oocytes using a combination of synchrotron Fourier transform-infrared and Raman spectroscopic mapping. , 2008, Analytical chemistry.

[24]  Dai Ping,et al.  Imaging of anticancer agent distribution by a slit-scanning Raman microscope , 2008, SPIE BiOS.

[25]  J. Greve,et al.  Localization study of Co-phthalocyanines in cells by Raman micro(spectro)scopy , 1999 .

[26]  S. Kazarian,et al.  Chemical Imaging of Live Cancer Cells in the Natural Aqueous Environment , 2009, Applied spectroscopy.

[27]  H. Robenek,et al.  Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis , 2006, Journal of Cell Science.

[28]  Ioan Notingher,et al.  Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro , 2006, Expert review of medical devices.

[29]  J. Polak,et al.  In situ Characterisation of Living Cells by Raman Spectroscopy , 2002 .

[30]  K. Brindle,et al.  Techniques: Visualizing apoptosis using nuclear magnetic resonance. , 2003, Trends in pharmacological sciences.

[31]  Ioan Notingher,et al.  In situ spectral monitoring of mRNA translation in embryonic stem cells during differentiation in vitro. , 2004, Analytical chemistry.

[32]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[33]  B Willekens,et al.  Nonresonant Raman imaging of protein distribution in single human cells. , 2003, Biopolymers.

[34]  Hongjie Dai,et al.  Multiplexed multicolor Raman imaging of live cells with isotopically modified single walled carbon nanotubes. , 2008, Journal of the American Chemical Society.

[35]  S. Mathur,et al.  Lipid nutrition and metabolism of cultured mammalian cells. , 1980, Progress in lipid research.

[36]  J. Greve,et al.  Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5 nm but not with 660 nm laser light. , 1991, Experimental cell research.

[37]  Alex Henderson,et al.  Spectral discrimination of live prostate and bladder cancer cell lines using Raman optical tweezers. , 2008, Journal of biomedical optics.

[38]  J Greve,et al.  Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells. , 2003, Biophysical journal.

[39]  Max Diem,et al.  Raman and Infrared Microspectral Imaging of Mitotic Cells , 2006, Applied spectroscopy.

[40]  L L Hench,et al.  Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells. , 2003, Biopolymers.

[41]  L L Hench,et al.  In situ monitoring of cell death using Raman microspectroscopy. , 2004, Biopolymers.

[42]  X. Xie,et al.  Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy Published, JLR Papers in Press, August 16, 2003. DOI 10.1194/jlr.D300022-JLR200 , 2003, Journal of Lipid Research.

[43]  Don McNaughton,et al.  Micro-Raman characterization of high- and low-spin heme moieties within single living erythrocytes. , 2002, Biopolymers.

[44]  Y. Kraan,et al.  Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[45]  L. Grochow,et al.  Effect of P-glycoprotein expression on the accumulation and cytotoxicity of topotecan (SK&F 104864), a new camptothecin analogue. , 1992, Cancer research.

[46]  Jonathan E. Schmitz,et al.  1H MRS‐visible lipids accumulate during apoptosis of lymphoma cells in vitro and in vivo , 2005, Magnetic resonance in medicine.

[47]  T. Cotter,et al.  Apoptosis and cancer: the genesis of a research field , 2009, Nature Reviews Cancer.

[48]  F F de Mul,et al.  Raman microspectroscopic approach to the study of human granulocytes. , 1991, Biophysical journal.

[49]  P. Lasch,et al.  FT-IR spectroscopic investigations of single cells on the subcellular level , 2002 .

[50]  Gavin Jell,et al.  In situ non‐invasive spectral discrimination between bone cell phenotypes used in tissue engineering , 2004, Journal of cellular biochemistry.

[51]  Christoph Krafft,et al.  Identification of organelles and vesicles in single cells by Raman microspectroscopic mapping , 2005 .

[52]  Rebecca C Taylor,et al.  Apoptosis: controlled demolition at the cellular level , 2008, Nature Reviews Molecular Cell Biology.

[53]  Gavin Jell,et al.  In vitro toxicology evaluation of pharmaceuticals using Raman micro‐spectroscopy , 2006, Journal of cellular biochemistry.

[54]  J. Greve,et al.  Studying single living cells and chromosomes by confocal Raman microspectroscopy , 1990, Nature.

[55]  A. Stringaro,et al.  1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells. , 2001, Biochimica et biophysica acta.

[56]  Christoph Krafft,et al.  Studies on stress-induced changes at the subcellular level by Raman microspectroscopic mapping. , 2006, Analytical chemistry.