Abstract

We present high-speed hyperspectral Raman imaging with integrated active-illumination for label-free compositional microanalysis. We show that high-quality Raman spectra can be acquired from as many as ~1,000 spots/sec semi-randomly distributed among a ~100x100 μm2 area without mechanical scanning. We demonstrate rapid data acquisition from three types of samples: 1) uniform, strong Raman scatterers, e.g., silicon substrates; 2) non-uniform, medium-strength Raman scatterers, e.g., polymer microparticles; and, 3) non-uniform, relatively weak Raman scatterers, e.g., bacterial spores. We compare the system performance to that of point-scan with an electron-multiplied CCD camera, as implemented in some commercial systems. The results suggest that our system not only provides significant imaging speed advantage for various types of samples, but also permits substantially longer integration time per spot, leading to superior signal-to-noise ratio data. Our system enables the rapid collection of high quality Raman spectra for reliable and robust compositional microanalysis that are potentially transformative in applications such as semiconductor material and device, polymer blend and biomedicine.

© 2013 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
    [CrossRef] [PubMed]
  2. C. Matthäus, S. Boydston-White, M. Miljković, M. Romeo, M. Diem, “Raman and infrared microspectral imaging of mitotic cells,” Appl. Spectrosc. 60(1), 1–8 (2006).
    [CrossRef] [PubMed]
  3. V. V. Pully, A. T. M. Lenferink, C. Otto, “Time-lapse Raman imaging of single live lymphocytes,” J. Raman Spectros. 42(2), 167–173 (2011).
    [CrossRef]
  4. B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
    [CrossRef] [PubMed]
  5. K. Kneipp, A. S. Haka, H. Kneipp, K. Badizadegan, N. Yoshizawa, C. Boone, K. E. Shafer-Peltier, J. T. Motz, R. R. Dasari, M. S. Feld, “Surface-enhanced Raman Spectroscopy in single living cells using gold nanoparticles,” Appl. Spectrosc. 56(2), 150–154 (2002).
    [CrossRef]
  6. T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
    [CrossRef] [PubMed]
  7. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
    [CrossRef] [PubMed]
  8. L. Gao, H. J. Zhou, M. J. Thrall, F. Li, Y. L. Yang, Z. Y. Wang, P. F. Luo, K. K. Wong, G. S. Palapattu, S. T. C. Wong, “Label-free high-resolution imaging of prostate glands and cavernous nerves using coherent anti-Stokes Raman scattering microscopy,” Biomed. Opt. Express 2(4), 915–926 (2011).
    [CrossRef] [PubMed]
  9. K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
    [CrossRef] [PubMed]
  10. J. Qi, W.-C. Shih, “Parallel Raman microspectroscopy using programmable multipoint illumination,” Opt. Lett. 37(8), 1289–1291 (2012).
    [CrossRef] [PubMed]
  11. S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
    [CrossRef]

2012 (1)

2011 (2)

2010 (1)

B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
[CrossRef] [PubMed]

2009 (2)

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

2008 (2)

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

2006 (1)

2002 (1)

1990 (1)

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Amiji, M.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Badizadegan, K.

Beier, B. D.

B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
[CrossRef] [PubMed]

Berger, A. J.

B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
[CrossRef] [PubMed]

Bohme, R.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Boone, C.

Boydston-White, S.

Chernenko, T.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

Dasari, R. R.

de Mul, F. F. M.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Diem, M.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

C. Matthäus, S. Boydston-White, M. Miljković, M. Romeo, M. Diem, “Raman and infrared microspectral imaging of mitotic cells,” Appl. Spectrosc. 60(1), 1–8 (2006).
[CrossRef] [PubMed]

Elschner, M.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Feld, M. S.

Freudiger, C. W.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Fujita, K.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

Gao, L.

Greve, J.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Haka, A. S.

Hamada, K.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

He, C. W.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Holtom, G. R.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Inouye, Y.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

Jovin, T. M.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Kang, J. X.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Kawata, S.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

Kneipp, H.

Kneipp, K.

Kobayashi, M.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

Lenferink, A. T. M.

V. V. Pully, A. T. M. Lenferink, C. Otto, “Time-lapse Raman imaging of single live lymphocytes,” J. Raman Spectros. 42(2), 167–173 (2011).
[CrossRef]

Li, F.

Lu, S.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Luo, P. F.

Matthäus, C.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

C. Matthäus, S. Boydston-White, M. Miljković, M. Romeo, M. Diem, “Raman and infrared microspectral imaging of mitotic cells,” Appl. Spectrosc. 60(1), 1–8 (2006).
[CrossRef] [PubMed]

Meisel, S.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Milane, L.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

Miljkovic, M.

Min, W.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Motz, J. T.

Otto, C.

V. V. Pully, A. T. M. Lenferink, C. Otto, “Time-lapse Raman imaging of single live lymphocytes,” J. Raman Spectros. 42(2), 167–173 (2011).
[CrossRef]

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Palapattu, G. S.

Popp, J.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Pully, V. V.

V. V. Pully, A. T. M. Lenferink, C. Otto, “Time-lapse Raman imaging of single live lymphocytes,” J. Raman Spectros. 42(2), 167–173 (2011).
[CrossRef]

Puppels, G. J.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Qi, J.

Quintero, L.

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

Quivey, R. G.

B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
[CrossRef] [PubMed]

Robert-Nicoud, M.

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Romeo, M.

Rosch, P.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Saar, B. G.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Shafer-Peltier, K. E.

Shih, W.-C.

Smith, N. I.

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

Stockel, S.

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Thrall, M. J.

Tsai, J. C.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Wang, Z. Y.

Wong, K. K.

Wong, S. T. C.

Xie, X. S.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Yang, Y. L.

Yoshizawa, N.

Zhou, H. J.

ACS Nano (1)

T. Chernenko, C. Matthäus, L. Milane, L. Quintero, M. Amiji, M. Diem, “Label-free Raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle systems,” ACS Nano 3(11), 3552–3559 (2009).
[CrossRef] [PubMed]

Appl. Spectrosc. (2)

Biomed. Opt. Express (1)

J. Biomed. Opt. (2)

K. Hamada, K. Fujita, N. I. Smith, M. Kobayashi, Y. Inouye, S. Kawata, “Raman microscopy for dynamic molecular imaging of living cells,” J. Biomed. Opt. 13(4), 044027 (2008).
[CrossRef] [PubMed]

B. D. Beier, R. G. Quivey, A. J. Berger, “Identification of different bacterial species in biofilms using confocal Raman microscopy,” J. Biomed. Opt. 15(6), 066001 (2010).
[CrossRef] [PubMed]

J. Raman Spectros. (2)

V. V. Pully, A. T. M. Lenferink, C. Otto, “Time-lapse Raman imaging of single live lymphocytes,” J. Raman Spectros. 42(2), 167–173 (2011).
[CrossRef]

S. Stockel, S. Meisel, R. Bohme, M. Elschner, P. Rosch, J. Popp, “Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy,” J. Raman Spectros. 40, 1469–1477 (2009).
[CrossRef]

Nature (1)

G. J. Puppels, F. F. M. de Mul, C. Otto, J. Greve, M. Robert-Nicoud, D. J. Arndt-Jovin, T. M. Jovin, “Studying single living cells and chromosomes by confocal Raman microspectroscopy,” Nature 347(6290), 301–303 (1990).
[CrossRef] [PubMed]

Opt. Lett. (1)

Science (1)

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. W. He, J. C. Tsai, J. X. Kang, X. S. Xie, “Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (4)

Fig. 1
Fig. 1

(a) System configuration; (b) image feature extraction, random pattern selection and hologram computation flow chart.

Fig. 2
Fig. 2

(a) Silicon image using the 520 cm−1 peak from 19 random illumination patterns within a 90x120 μm2 field of view; (b) Measured peak positions vs. laser spot x position using seven acetaminophen Raman peaks and one silicon Raman peak. These curves enable the correct assignment of Raman shifts for each laser excitation spot; (c), (d) and (e) silicon spectra near 520 cm−1 peak acquired by EMCCD with 1 and 10 ms integration time, and low-noise CCD with 25 ms integration time, respectively.

Fig. 3
Fig. 3

(a) Bright-field image of a mixed sample of 2 μm PS and MR microspheres; (b) binarized centroid image after image processing; (c) random selection of 15% of microspheres and splitting into four color-coded groups (patterns); (d-g) holograms corresponding to the four groups (patterns) selected in (c); (h-k) raw Raman images corresponding to the illumination patterns according to the holograms in (d-g).

Fig. 4
Fig. 4

(a) Classification of polymer microparticles with PS marked by blue circles and MR red circles; (b) Raman spectra of PS and MR with major peaks used in classification; (c, top image) three microparticles of interest are illuminated by three laser spots in the windowed region in pattern #3, or Fig. 3(j); (c, bottom spectra) Raman spectra from the nine CCD rows corresponding to the same region in the top image; (d) bright-field image of spores and 4 color-coded sub-groups for 4 illumination patterns; (e) corresponding Raman image by 4 illumination patterns; (f) representative spore Raman spectra with characteristic peaks marked by *.

Metrics