Abstract

While Raman hyperspectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: (1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; and (2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples, we show that Raman hyperspectral data sets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins, and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering.

© 2017 The Author(s)

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  2. B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.
  3. G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.
  4. C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.
  5. K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.
  6. L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.
  7. R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.
  8. N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.
  9. A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.
  10. M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.
  11. K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.
  12. H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.
  13. F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.
  14. A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.
  15. S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.
  16. S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.
  17. H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.
  18. H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.
  19. L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.
  20. K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.
  21. K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.
  22. L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.
  23. F. Sinjab, K. Kong, G. Gibson, S. Varma, and et al., “Tissue Diagnosis Using Power-Sharing Multifocal Raman Micro-Spectroscopy and Auto-Fluorescence Imaging”Biomed. Opt. Express . 2016.  7(8): 2993–3006.
  24. C.A. Lieber and A. Mahadevan-Jansen, “Automated Method for Subtraction of Fluorescence from Biological Raman Spectra”. Appl. Spectrosc . 2003.  57(11): 1363–1367.
  25. B.D. Beier and A.J. Berger, “Method for Automated Background Subtraction from Raman Spectra Containing Known Contaminants”. Analyst . 2009.  134(6): 1198–1202.
  26. N.J. Everall, “Modeling and Measuring the Effect of Refraction on the Depth Resolution of Confocal Raman Microscopy”. Appl. Spectrosc . 2000.  54(6): 773–782.
  27. N.J. Everall, “Confocal Raman Microscopy: Why the Depth Resolution and Spatial Accuracy Can Be Much Worse Than You Think”. Appl. Spectrosc . 2000.  54(10): 1515–1520.
  28. K.E. Gascoigne and S.E. Taylor, “How Do Anti-Mitotic Drugs Kill Cancer Cells?”. J. Cell Sci . 2009.  122: 2579–2585.

2016 (5)

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

F. Sinjab, K. Kong, G. Gibson, S. Varma, and et al., “Tissue Diagnosis Using Power-Sharing Multifocal Raman Micro-Spectroscopy and Auto-Fluorescence Imaging”Biomed. Opt. Express . 2016.  7(8): 2993–3006.

2015 (4)

L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

2014 (1)

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

2013 (1)

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

2012 (2)

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

2011 (2)

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

2010 (2)

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.

2009 (2)

K.E. Gascoigne and S.E. Taylor, “How Do Anti-Mitotic Drugs Kill Cancer Cells?”. J. Cell Sci . 2009.  122: 2579–2585.

B.D. Beier and A.J. Berger, “Method for Automated Background Subtraction from Raman Spectra Containing Known Contaminants”. Analyst . 2009.  134(6): 1198–1202.

2008 (1)

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

2003 (3)

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

C.A. Lieber and A. Mahadevan-Jansen, “Automated Method for Subtraction of Fluorescence from Biological Raman Spectra”. Appl. Spectrosc . 2003.  57(11): 1363–1367.

2002 (1)

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

2000 (2)

N.J. Everall, “Modeling and Measuring the Effect of Refraction on the Depth Resolution of Confocal Raman Microscopy”. Appl. Spectrosc . 2000.  54(6): 773–782.

N.J. Everall, “Confocal Raman Microscopy: Why the Depth Resolution and Spatial Accuracy Can Be Much Worse Than You Think”. Appl. Spectrosc . 2000.  54(10): 1515–1520.

1990 (1)

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Ashton, L.

L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.

Baranska, M.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

Beier, B.D.

B.D. Beier and A.J. Berger, “Method for Automated Background Subtraction from Raman Spectra Containing Known Contaminants”. Analyst . 2009.  134(6): 1198–1202.

Berger, A.J.

B.D. Beier and A.J. Berger, “Method for Automated Background Subtraction from Raman Spectra Containing Known Contaminants”. Analyst . 2009.  134(6): 1198–1202.

Boitor, I.

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

Boitor, R.

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

Buttery, L.D.

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

Byrne, H.J.

L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.

Caron, N.J.

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

Chun, Y.W.

L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.

Clarke, N.M.

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

Czamara, K.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

De Mul, F.F.M.

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Derely, L.

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

Durand, J.C.

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

El-Mashtoly, S.F.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

Elsheikha, R.A.

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

Endo, H.

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

Everall, N.J.

N.J. Everall, “Modeling and Measuring the Effect of Refraction on the Depth Resolution of Confocal Raman Microscopy”. Appl. Spectrosc . 2000.  54(6): 773–782.

N.J. Everall, “Confocal Raman Microscopy: Why the Depth Resolution and Spatial Accuracy Can Be Much Worse Than You Think”. Appl. Spectrosc . 2000.  54(10): 1515–1520.

Fujita, K.

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

Funk, R.H.

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

Galler, U.

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

Gascoigne, K.E.

K.E. Gascoigne and S.E. Taylor, “How Do Anti-Mitotic Drugs Kill Cancer Cells?”. J. Cell Sci . 2009.  122: 2579–2585.

Ghita, A.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

Gibson, G.

Glaser, T.

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

Goh, T.H.

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

Goodacre, R.

L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.

Greve, J.

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Hamada, K.

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

Henkel,

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

Hennelly, B.M.

L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.

Hollywood, K.A.

L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.

Kaczor, A.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

Kann, B.

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

Kerr, L.T.

L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.

Knetschke, T.

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

Kobayashi, M.

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

Kong, K.

Konorov, S.O.

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

Kraan, Y.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

Krafft, C.

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

Lenferink, A.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

Lieber, C.A.

C.A. Lieber and A. Mahadevan-Jansen, “Automated Method for Subtraction of Fluorescence from Biological Raman Spectra”. Appl. Spectrosc . 2003.  57(11): 1363–1367.

Mahadevan-Jansen, A.

C.A. Lieber and A. Mahadevan-Jansen, “Automated Method for Subtraction of Fluorescence from Biological Raman Spectra”. Appl. Spectrosc . 2003.  57(11): 1363–1367.

Mather, M.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

Mavarani, L.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

Mosig, A.

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

Naemat, H.M.

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

Notingher,

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

Notingher, I.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

Offerhaus, H.L.

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

Okada, M.

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

Okubo, Y.

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

Otto, C.

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Palonpon, A.F.

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

Partridge, K.

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

Pascut, F.C.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

Patel, P.

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

Petersen, D.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

Petko, F.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

Piret, J.M.

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

Puppels, G.J.

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Ramoji, K.

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

Salehi, H.

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

Schulze, H.G.

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

Siegner, A.

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

Sinjab, F.

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

F. Sinjab, K. Kong, G. Gibson, S. Varma, and et al., “Tissue Diagnosis Using Power-Sharing Multifocal Raman Micro-Spectroscopy and Auto-Fluorescence Imaging”Biomed. Opt. Express . 2016.  7(8): 2993–3006.

Smith, N.I.

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

Sottile, V.

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

Strohbuecker, S.

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

Taylor, S.E.

K.E. Gascoigne and S.E. Taylor, “How Do Anti-Mitotic Drugs Kill Cancer Cells?”. J. Cell Sci . 2009.  122: 2579–2585.

Uzunbajakava, N.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

Varma, S.

Vegh, A.G.

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

Volokhina, E.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

Webster, T.J.

L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.

Welch, N.

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

Windbergs, M.

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

Yang, X.

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

Yosef, H.K.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

Zhang, L.

L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.

Zoladek, A.

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

Adv. Drug Delivery Rev (1)

B. Kann, H.L. Offerhaus, M. Windbergs, and C. Otto, “Raman Microscopy for Cellular Investigations: From Single Cell Imaging to Drug Carrier Uptake Visualization”. Adv. Drug Delivery Rev . 2015.  89: 71–90.

Anal. Chem (3)

H.G. Schulze, S.O. Konorov, N.J. Caron, J.M. Piret, and et al., “Assessing Differentiation Status of Human Embryonic Stem Cells Noninvasively Using Raman Microspectroscopy”Anal. Chem . 2010.  82(12): 5020–5027.

A. Ghita, F.C. Pascut, M. Mather, V. Sottile, and I. Notingher, “Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status”. Anal. Chem . 2012.  84(7): 3155–3162.

S.F. El-Mashtoly, H.K. Yosef, D. Petersen, L. Mavarani, and et al., “Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells”Anal. Chem . 2015.  87(14): 7297–7304.

Anal. Methods (1)

L.T. Kerr, H.J. Byrne, and B.M. Hennelly, “Optimal Choice of Sample Substrate and Laser Wavelength for Raman Spectroscopic Analysis of Biological Specimen”. Anal. Methods . 2015.  7(12): 5041–5052.

Analyst (4)

S.F. El-Mashtoly, D. Petersen, H.K. Yosef, A. Mosig, and et al., “Label-Free Imaging of Drug Distribution and Metabolism in Colon Cancer Cells by Raman Microscopy”Analyst . 2014.  139(5): 1155–1161.

L. Ashton, K.A. Hollywood, and R. Goodacre, “Making Colourful Sense of Raman Images of Single Cells”. Analyst . 2015.  140(6): 1852–1858.

K. Czamara, F. Petko, M. Baranska, and A. Kaczor, “Raman Microscopy at the Subcellular Level: a Study on Early Apoptosis in Endothelial Cells Induced by Fas Ligand and Cycloheximide”. Analyst . 2016.  141(4): 1390–1397.

B.D. Beier and A.J. Berger, “Method for Automated Background Subtraction from Raman Spectra Containing Known Contaminants”. Analyst . 2009.  134(6): 1198–1202.

Appl. Phys. Lett (1)

H. Salehi, L. Derely, A.G. Vegh, J.C. Durand, and et al., “Label-Free Detection of Anticancer Drug Paclitaxel in Living Cells by Confocal Raman Microscopy”Appl. Phys. Lett . 2013.  102(11): 113701.

Appl. Spectrosc (3)

N.J. Everall, “Modeling and Measuring the Effect of Refraction on the Depth Resolution of Confocal Raman Microscopy”. Appl. Spectrosc . 2000.  54(6): 773–782.

N.J. Everall, “Confocal Raman Microscopy: Why the Depth Resolution and Spatial Accuracy Can Be Much Worse Than You Think”. Appl. Spectrosc . 2000.  54(10): 1515–1520.

C.A. Lieber and A. Mahadevan-Jansen, “Automated Method for Subtraction of Fluorescence from Biological Raman Spectra”. Appl. Spectrosc . 2003.  57(11): 1363–1367.

Biochem. Biophys. Res. Commun (1)

K. Partridge, X. Yang, N.M. Clarke, Y. Okubo, and et al., “Adenoviral BMP-2 Gene Transfer in Mesenchymal Stem Cells: In Vitro And In Vivo Bone Formation on Biodegradable Polymer Scaffolds”Biochem. Biophys. Res. Commun . 2002.  292(1): 144–152.

Biomed. Opt. Express (1)

Biophys. J (2)

F.C. Pascut, T.H. Goh, N. Welch, L.D. Buttery, and et al., “Noninvasive Detection and Imaging of Molecular Markers in Live Cardiomyocytes Derived from Human Embryonic Stem Cells”Biophys. J . 2011.  100(1): 251–259.

N. Uzunbajakava, A. Lenferink, Y. Kraan, E. Volokhina, and et al., “Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells”Biophys. J . 2003.  84(6): 3968–3981.

Faraday Discuss (1)

R. Boitor, F. Sinjab, S. Strohbuecker, V. Sottile, and et al., “Towards Quantitative Molecular Mapping of Cells by Raman Microscopy: Using AFM for Decoupling Molecular Concentration and Cell Topography”Faraday Discuss . 2016.  187: 199–212.

Int. J. Nanomed (1)

L. Zhang, Y.W. Chun, and T.J. Webster, “Decreased Lung Carcinoma Cell Density on Select Polymer Nanometer Surface Features for Lung Replacement Therapies”. Int. J. Nanomed . 2010.  5: 269–275.

J. Biomed. Opt (1)

K. Hamada, K. Fujita, N.I. Smith, M. Kobayashi, and et al., “Raman Microscopy for Dynamic Molecular Imaging of Living Cells”J. Biomed. Opt . 2008.  13(4): 044027.

J. Cell Sci (1)

K.E. Gascoigne and S.E. Taylor, “How Do Anti-Mitotic Drugs Kill Cancer Cells?”. J. Cell Sci . 2009.  122: 2579–2585.

J. Raman Spectrosc (2)

A. Zoladek, F.C. Pascut, P. Patel, and I. Notingher, “Non‐Invasive Time‐Course Imaging of Apoptotic Cells by Confocal Raman Micro‐Spectroscopy”. J. Raman Spectrosc . 2011.  42(3): 251–258.

K. Ramoji, U. Galler, T. Glaser, Henkel, and et al., “Characterization of Different Substrates for Raman Spectroscopic Imaging of Eukaryotic Cells”J. Raman Spectrosc . 2016.  47(7): 773–786.

Nature (1)

G.J. Puppels, F.F.M. De Mul, C. Otto, J. Greve, and et al., “Studying Single Living Cells and Chromosomes by Confocal Raman Microspectroscopy”Nature . 1990.  347(6290): 301.

Proc. Natl. Acad. Sci. U. S. A (1)

M. Okada, N.I. Smith, A.F. Palonpon, H. Endo, and et al., “Label-Free Raman Observation of Cytochrome c Dynamics During Apoptosis”Proc. Natl. Acad. Sci. U. S. A . 2012.  109(1): 28–32.

Sci. Rep (1)

H.M. Naemat, R.A. Elsheikha, I. Boitor, and Notingher, . “Tracing Amino Acid Exchange During Host-Pathogen Interaction by Combined Stable-Isotope Time-Resolved Raman Spectral Imaging”. Sci. Rep . 2016.  6: 20811.

Vib. Spectrosc (1)

C. Krafft, T. Knetschke, A. Siegner, R.H. Funk, and et al., “Mapping of Single Cells by Near Infrared Raman Microspectroscopy”Vib. Spectrosc . 2003.  32(1): 75–83.

Other (1)

D.W. Shipp, F. Sinjab, I. Notingher. “Raman spectroscopy: Techniques and Applications in the Life Sciences”. Adv. Opt. Photonics. 2017. 9(2): 315–428.

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