Abstract

We propose and demonstrate a dark-field imaging technique capable of automated identification of individual bacteria. An 87-channel multispectral system capable of angular and spectral resolution was used to measure the scattering spectrum of various bacteria in culture smears. Spectra were compared between various species and between various preparations of the same species. A 15-channel system was then used to prove the viability of bacterial identification with a relatively simple microscope system. A simple classifier was able to identify four of six bacterial species with greater than 90% accuracy in bacteria-by-bacteria testing.

© 2013 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. International Union against Tuberculosis and Disease, “Priorities for tuberculosis bacteriology services in low income countries,” (2007).
  2. The New Diagnostics Working Group of the Stop TB Partnership, “Pathways to better diagnostics for tuberculosis” (2009).
  3. P. J. Wyatt, “Differential light scattering: a physical method for identifying living bacterial cells,” Appl. Opt. 7(10), 1879–1896 (1968).
    [CrossRef] [PubMed]
  4. P. J. Wyatt, D. T. Phillips, “Structure of single bacteria from light scattering,” J. Theor. Biol. 37(3), 493–501 (1972).
    [CrossRef] [PubMed]
  5. M. Bartholdi, G. C. Salzman, R. D. Hiebert, M. Kerker, “Differential light scattering photometer for rapid analysis of single particles in flow,” Appl. Opt. 19(10), 1573–1581 (1980).
    [CrossRef] [PubMed]
  6. M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
    [CrossRef] [PubMed]
  7. M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
    [CrossRef] [PubMed]
  8. P. F. Mullaney, P. N. Dean, “Cell sizing: a small-angle light-scattering method for sizing particles of low relative refractive index,” Appl. Opt. 8(11), 2361–2362 (1969).
    [CrossRef] [PubMed]
  9. V. J. Morris, B. R. Jennings, “Light scattering by bacteria. I. Angular dependence of the scattered intensity,” Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 338(1613), 197–208 (1974).
    [CrossRef]
  10. B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
    [CrossRef] [PubMed]
  11. G. A. Jamjoom, “Dark-field microscopy for detection of malaria in unstained blood films,” J. Clin. Microbiol. 17(5), 717–721 (1983).
    [PubMed]
  12. B. K. Wilson, M. R. Behrend, M. P. Horning, M. C. Hegg, “Detection of malarial byproduct hemozoin utilizing its unique scattering properties,” Opt. Express 19(13), 12190–12196 (2011).
    [CrossRef] [PubMed]
  13. S. A. Larsen, B. M. Steiner, A. H. Rudolph, S. A. Larsen, and B. M. Steiner, “Laboratory diagnosis and interpretation of tests for syphilis,” Clin. Microbio. Rev. 8, (1995).
  14. R. M. Macnab, “Examination of bacterial flagellation by dark-field examination of bacterial flagellation by dark-field microscopy,” J. Clin. Microbiol.4, (1976).
  15. K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
    [CrossRef] [PubMed]
  16. J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
    [CrossRef] [PubMed]
  17. J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
    [CrossRef] [PubMed]
  18. A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
    [CrossRef] [PubMed]
  19. P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
    [PubMed]
  20. UNICEF, WHO, “Pneumonia: the forgotten killer of children,” (2006).
  21. M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
    [CrossRef]
  22. P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
    [PubMed]
  23. R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing using MATLAB: 2nd Ed. (Gatesmark Publishing 2009), Chap, 13.
  24. H. F. Grahan and P. Geladi, Techniques and Applications of Hyperspectral Image Analysis (John Wiley and Sons, 2007).

2011 (3)

B. K. Wilson, M. R. Behrend, M. P. Horning, M. C. Hegg, “Detection of malarial byproduct hemozoin utilizing its unique scattering properties,” Opt. Express 19(13), 12190–12196 (2011).
[CrossRef] [PubMed]

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

2009 (1)

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

2008 (2)

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

2007 (1)

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

2004 (1)

M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
[CrossRef]

1983 (1)

G. A. Jamjoom, “Dark-field microscopy for detection of malaria in unstained blood films,” J. Clin. Microbiol. 17(5), 717–721 (1983).
[PubMed]

1980 (1)

1979 (1)

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

1976 (1)

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
[CrossRef] [PubMed]

1974 (1)

V. J. Morris, B. R. Jennings, “Light scattering by bacteria. I. Angular dependence of the scattered intensity,” Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 338(1613), 197–208 (1974).
[CrossRef]

1972 (1)

P. J. Wyatt, D. T. Phillips, “Structure of single bacteria from light scattering,” J. Theor. Biol. 37(3), 493–501 (1972).
[CrossRef] [PubMed]

1969 (2)

P. F. Mullaney, P. N. Dean, “Cell sizing: a small-angle light-scattering method for sizing particles of low relative refractive index,” Appl. Opt. 8(11), 2361–2362 (1969).
[CrossRef] [PubMed]

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

1968 (1)

Aaron, J.

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

Banada, P. P.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Bartholdi, M.

Beg, M. F.

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

Behrend, M. R.

Bharadwa, P.

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

Chew, H.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Chilkoti, A.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Comstock, G. W.

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

Cooke, D. D.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Cristóbal, G.

M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
[CrossRef]

Crow, M. J.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Curtis, J. L.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Dean, P. N.

Erb-Downward, J. R.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Felton, F. G.

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

Forero, M. G.

M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
[CrossRef]

Freeman, C. M.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Han, M. K.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Harrison, N.

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

Hegg, M. C.

Herzenberg, L. A.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
[CrossRef] [PubMed]

Hiebert, R. D.

Hill, S. L.

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

Hirleman, E. D.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Horning, M. P.

Huffnagle, G. B.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Jamjoom, G. A.

G. A. Jamjoom, “Dark-field microscopy for detection of malaria in unstained blood films,” J. Clin. Microbiol. 17(5), 717–721 (1983).
[PubMed]

Jennings, B. R.

V. J. Morris, B. R. Jennings, “Light scattering by bacteria. I. Angular dependence of the scattered intensity,” Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 338(1613), 197–208 (1974).
[CrossRef]

Kerker, M.

M. Bartholdi, G. C. Salzman, R. D. Hiebert, M. Kerker, “Differential light scattering photometer for rapid analysis of single particles in flow,” Appl. Opt. 19(10), 1573–1581 (1980).
[CrossRef] [PubMed]

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Kratohvil, J. P.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Lary, T.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Lee, M. P.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Loken, M. R.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
[CrossRef] [PubMed]

Macnab, R. M.

R. M. Macnab, “Examination of bacterial flagellation by dark-field examination of bacterial flagellation by dark-field microscopy,” J. Clin. Microbiol.4, (1976).

Marinakos, S.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Martinez, F. J.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

McCloskey, L.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

McNulty, P. J.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Monroe, P. W.

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

Morris, V. J.

V. J. Morris, B. R. Jennings, “Light scattering by bacteria. I. Angular dependence of the scattered intensity,” Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 338(1613), 197–208 (1974).
[CrossRef]

Muchmore, H. G.

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

Mullaney, P. F.

Ostrander, J.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Phillips, D. T.

P. J. Wyatt, D. T. Phillips, “Structure of single bacteria from light scattering,” J. Theor. Biol. 37(3), 493–501 (1972).
[CrossRef] [PubMed]

Pirtle, J. K.

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

Pye, A.

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

Ragheb, K.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Rajwa, B.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Rao, J.

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

Robinson, J. P.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Sadaphal, P.

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

Salzman, G. C.

Schmidt, L. A.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Sculley, M.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

Seekell, K.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Sokolov, K.

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

Sroubek, F.

M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
[CrossRef]

Stockley, R. A.

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

Sundaram, B.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Sweet, R. G.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
[CrossRef] [PubMed]

Thompson, D. L.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Toews, G. B.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Travis, K.

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

Venkatapathi, M.

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Wax, A.

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

Wilson, B. K.

Wyatt, P. J.

P. J. Wyatt, D. T. Phillips, “Structure of single bacteria from light scattering,” J. Theor. Biol. 37(3), 493–501 (1972).
[CrossRef] [PubMed]

P. J. Wyatt, “Differential light scattering: a physical method for identifying living bacterial cells,” Appl. Opt. 7(10), 1879–1896 (1968).
[CrossRef] [PubMed]

Young, V. B.

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Appl. Microbiol. (1)

P. W. Monroe, H. G. Muchmore, F. G. Felton, J. K. Pirtle, “Quantitation of microorganisms in sputum,” Appl. Microbiol. 18(2), 214–220 (1969).
[PubMed]

Appl. Opt. (3)

Cytometry A (1)

B. Rajwa, M. Venkatapathi, K. Ragheb, P. P. Banada, E. D. Hirleman, T. Lary, J. P. Robinson, “Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier,” Cytometry A 73(4), 369–379 (2008).
[CrossRef] [PubMed]

Int. J. Tuberc. Lung Dis. (1)

P. Sadaphal, J. Rao, G. W. Comstock, M. F. Beg, “Image processing techniques for identifying Mycobacterium tuberculosis in Ziehl-Neelsen stains,” Int. J. Tuberc. Lung Dis. 12(5), 579–582 (2008).
[PubMed]

J. Biomed. Opt. (1)

K. Seekell, M. J. Crow, S. Marinakos, J. Ostrander, A. Chilkoti, A. Wax, “Hyperspectral molecular imaging of multiple receptors using immunolabeled plasmonic nanoparticles,” J. Biomed. Opt. 16(11), 116003 (2011).
[CrossRef] [PubMed]

J. Clin. Microbiol. (1)

G. A. Jamjoom, “Dark-field microscopy for detection of malaria in unstained blood films,” J. Clin. Microbiol. 17(5), 717–721 (1983).
[PubMed]

J. Clin. Pathol. (1)

A. Pye, S. L. Hill, P. Bharadwa, R. A. Stockley, “Effect of storage and postage on recovery and quantitation of bacteria in sputum samples,” J. Clin. Pathol. 61(3), 352–354 (2007).
[CrossRef] [PubMed]

J. Histochem. Cytochem. (2)

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24(1), 284–291 (1976).
[CrossRef] [PubMed]

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M. P. Lee, “Light scattering and fluorescence by small particles having internal structure,” J. Histochem. Cytochem. 27(1), 250–263 (1979).
[CrossRef] [PubMed]

J. Theor. Biol. (1)

P. J. Wyatt, D. T. Phillips, “Structure of single bacteria from light scattering,” J. Theor. Biol. 37(3), 493–501 (1972).
[CrossRef] [PubMed]

Nano Lett. (1)

J. Aaron, K. Travis, N. Harrison, K. Sokolov, “Dynamic imaging of molecular assemblies in live cells based on nanoparticle plasmon resonance coupling,” Nano Lett. 9(10), 3612–3618 (2009).
[CrossRef] [PubMed]

Opt. Express (1)

PLoS ONE (1)

J. R. Erb-Downward, D. L. Thompson, M. K. Han, C. M. Freeman, L. McCloskey, L. A. Schmidt, V. B. Young, G. B. Toews, J. L. Curtis, B. Sundaram, F. J. Martinez, G. B. Huffnagle, “Analysis of the lung microbiome in the “healthy” smoker and in COPD,” PLoS ONE 6(2), e16384 (2011).
[CrossRef] [PubMed]

Proc. Roy. Soc. A: Math. Phys. Eng. Sci. (1)

V. J. Morris, B. R. Jennings, “Light scattering by bacteria. I. Angular dependence of the scattered intensity,” Proc. Roy. Soc. A: Math. Phys. Eng. Sci. 338(1613), 197–208 (1974).
[CrossRef]

Real-Time Imag. (1)

M. G. Forero, F. Sroubek, G. Cristóbal, “Identification of tuberculosis bacteria based on shape and color,” Real-Time Imag. 10(4), 251–262 (2004).
[CrossRef]

Other (7)

R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing using MATLAB: 2nd Ed. (Gatesmark Publishing 2009), Chap, 13.

H. F. Grahan and P. Geladi, Techniques and Applications of Hyperspectral Image Analysis (John Wiley and Sons, 2007).

International Union against Tuberculosis and Disease, “Priorities for tuberculosis bacteriology services in low income countries,” (2007).

The New Diagnostics Working Group of the Stop TB Partnership, “Pathways to better diagnostics for tuberculosis” (2009).

UNICEF, WHO, “Pneumonia: the forgotten killer of children,” (2006).

S. A. Larsen, B. M. Steiner, A. H. Rudolph, S. A. Larsen, and B. M. Steiner, “Laboratory diagnosis and interpretation of tests for syphilis,” Clin. Microbio. Rev. 8, (1995).

R. M. Macnab, “Examination of bacterial flagellation by dark-field examination of bacterial flagellation by dark-field microscopy,” J. Clin. Microbiol.4, (1976).

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 (8)

Fig. 1
Fig. 1

(a) Theoretical collection efficiencies of the three rings of the VDA. The black outline is the unpolarized scattering intensity of a 2 µm silica sphere in cytoseal at 550 nm. (b) Schematic for the 87-channel MARDI microscope. Mchrm. = monochromator, VDA = variable dark-field aperture, Obj. = objective.

Fig. 2
Fig. 2

Diagram of MARDI post processing and classification algorithm.

Fig. 3
Fig. 3

Sample MARDI images of M. smegmatis (a-c) and S. aureus (d-f). (a) and (d) show a portion of a single channel without post processing. Each frame is 80x80 µm. (b) and (e) show the same frames colored by assigning RGB values to pixel-by-pixel PCA score for three modes. (c) and (f) show the same field as (a) and (d), respectively, with object identification and summing prior to PCA. RGB values are assigned to three PCA mode scores. In all cases mode scores were chosen to maximize the visible difference between species.

Fig. 4
Fig. 4

Principal components score plots for various intra-species tests. (a) M. smegmatis prepared with method C6, C12, C24, and C48, (b) S. aureas from purchased slides and slides prepared from lab culture, (c) M. smegmatis prepared with methods N, C24, and SP.

Fig. 5
Fig. 5

(a) Principal components score plot for two samples each of normally prepared and membrane stripped P. aeruginosa. (b, c) Micrographs of Gram stained P. aeruginosa without (b) and with (c) membrane stripped.

Fig. 6
Fig. 6

Scattering spectra of the eight tested bacteria species. The three dark-field aperture setting are denoted by colors corresponding to Fig. 1(b) (ring 1 = blue, ring 2 = green, ring 3 = red). Error bars show the standard error of the mean for sample sizes ranging from N = 346 for S. pneumoniae to N = 2172 for P. aeruginosa.

Fig. 7
Fig. 7

Principal components score plots for various inter-species tests. (a) H. influenzae and S. aureas. (b) two preparations of M. smegmatis versus M. bovis BCG.

Fig. 8
Fig. 8

(a) PCA scores for four species scanned using the 15 channel system, showing good cluster separation. (b) Sensitivity for classification of different species as a function of added Gaussian noise.

Tables (4)

Tables Icon

Table 1 Classifier results for culture age test with M. smegmatis

Tables Icon

Table 2 Classifier results for culture preparation tests of M. smegmatis

Tables Icon

Table 3 Classifier results for interspecies tests with 87 channel system

Tables Icon

Table 4 Classifier results for interspecies tests with 15 channel system

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

d j ( x ) = ln P ( w j ) 1 2 ln | C j | 1 2 [ ( x v j ) T C j ( x v j ) ] ,

Metrics