Abstract

Efficient image classification of microscopic fluorescent spheres is demonstrated with a supervised backpropagation neural network (NN) that uses as inputs the major color histogram representation of the fluorescent image to be classified. Two techniques are tested for the major color search: (1) cluster mean (CM) and (2) Kohonen’s self-organizing feature map (SOFM). The method is shown to have higher recognition rates than Swain and Ballard’s Color Indexing by histogram intersection. Classification with SOFM-generated histograms as inputs to the classifier NN achieved the best recognition rate (90%) for cases of normal, scaled, defocused, photobleached, and combined images of AMCA (7-Amino-4-Methylcoumarin-3-Acetic Acid) and FITC (Fluorescein Isothiocynate)-stained microspheres.

© 2001 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).
  2. M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
    [Crossref] [PubMed]
  3. E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
    [Crossref] [PubMed]
  4. S. Abrams, “Fluorescent Markers: GFP Joins the Common Dyes,” Biophotonics International 5, pp 48–54 (March/April 1998).
  5. C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
    [Crossref]
  6. C. Palmes-Saloma and C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology 131, 56–66 (2000).
    [Crossref]
  7. M. Swain and D. Ballard, “Color indexing,” International J. Computer Vision 7, 11–32 (1991).
    [Crossref]
  8. B. Funt and G. Finlayson, “Color constant color indexing,” IEEE Trans Pattern Analysis Machine Intelligence 17, 522–529 (1995).
    [Crossref]
  9. D. Slater and G. Healey, “Global color constancy: recognition of objects by use of illumination-invariant properties of color distributions,” J. Opt. Soc. Am. A 11, 3003–3010 (1994).
    [Crossref]
  10. P. Ennesser and G. Medioni, “Finding Waldo, or focus of attention using local color information,” IEEE Trans. Pattern Analysis and Machine Intelligence 17, 805–809 (1993).
    [Crossref]
  11. B. Mel, “SEEMORE: Combining color, shape, and texture histogramming in a neurally inspired approach to visual object recognition,” Neural Computation 9, 777–804 (1997).
    [Crossref] [PubMed]
  12. J. Lampinen and S. Smolander, “Self-organizing feature extraction in recognition of wood surface defects and color images,” Intl. J. Pattern Recognition and Artificial Intelligence,  10, 97–113 (1996).
    [Crossref]
  13. C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
    [Crossref]
  14. B.-L. Yeo and B. Liu. “Rapid scene analysis on compressed video,” IEEE Trans. Circuits and Systems for Video Technology 5, 533–544 (1995).
    [Crossref]
  15. S. Inoue, Video Microscopy, (Plenum Press, New York1986).
  16. B. Herman, Fluorescence Microscopy2nd Ed. (Springer-Verlag, Singapore1998).
  17. M. Soriano and C. Saloma, “Improved classification robustness for noisy cell images represented as principal-component projections in a hybrid recognition system,” Appl. Opt. 37, 3628–3838 (1998).
    [Crossref]

2000 (1)

C. Palmes-Saloma and C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology 131, 56–66 (2000).
[Crossref]

1998 (3)

S. Abrams, “Fluorescent Markers: GFP Joins the Common Dyes,” Biophotonics International 5, pp 48–54 (March/April 1998).

C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
[Crossref]

M. Soriano and C. Saloma, “Improved classification robustness for noisy cell images represented as principal-component projections in a hybrid recognition system,” Appl. Opt. 37, 3628–3838 (1998).
[Crossref]

1997 (1)

B. Mel, “SEEMORE: Combining color, shape, and texture histogramming in a neurally inspired approach to visual object recognition,” Neural Computation 9, 777–804 (1997).
[Crossref] [PubMed]

1996 (3)

J. Lampinen and S. Smolander, “Self-organizing feature extraction in recognition of wood surface defects and color images,” Intl. J. Pattern Recognition and Artificial Intelligence,  10, 97–113 (1996).
[Crossref]

M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
[Crossref] [PubMed]

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

1995 (2)

B. Funt and G. Finlayson, “Color constant color indexing,” IEEE Trans Pattern Analysis Machine Intelligence 17, 522–529 (1995).
[Crossref]

B.-L. Yeo and B. Liu. “Rapid scene analysis on compressed video,” IEEE Trans. Circuits and Systems for Video Technology 5, 533–544 (1995).
[Crossref]

1994 (2)

C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
[Crossref]

D. Slater and G. Healey, “Global color constancy: recognition of objects by use of illumination-invariant properties of color distributions,” J. Opt. Soc. Am. A 11, 3003–3010 (1994).
[Crossref]

1993 (1)

P. Ennesser and G. Medioni, “Finding Waldo, or focus of attention using local color information,” IEEE Trans. Pattern Analysis and Machine Intelligence 17, 805–809 (1993).
[Crossref]

1991 (1)

M. Swain and D. Ballard, “Color indexing,” International J. Computer Vision 7, 11–32 (1991).
[Crossref]

1990 (1)

S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).

Abrams, S.

S. Abrams, “Fluorescent Markers: GFP Joins the Common Dyes,” Biophotonics International 5, pp 48–54 (March/April 1998).

Andersson-Engels, S.

S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).

Ballard, D.

M. Swain and D. Ballard, “Color indexing,” International J. Computer Vision 7, 11–32 (1991).
[Crossref]

Bar-Am, I.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

du Manoir, S.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Ennesser, P.

P. Ennesser and G. Medioni, “Finding Waldo, or focus of attention using local color information,” IEEE Trans. Pattern Analysis and Machine Intelligence 17, 805–809 (1993).
[Crossref]

Equitz, W.

C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
[Crossref]

Faloutsos, C.

C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
[Crossref]

Ferguson-Smith, M.A.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Finlayson, G.

B. Funt and G. Finlayson, “Color constant color indexing,” IEEE Trans Pattern Analysis Machine Intelligence 17, 522–529 (1995).
[Crossref]

Flickner, M.

C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
[Crossref]

Funt, B.

B. Funt and G. Finlayson, “Color constant color indexing,” IEEE Trans Pattern Analysis Machine Intelligence 17, 522–529 (1995).
[Crossref]

Garini, Y.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Gwyn Ballard, S.

M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
[Crossref] [PubMed]

Healey, G.

Herman, B.

B. Herman, Fluorescence Microscopy2nd Ed. (Springer-Verlag, Singapore1998).

Inoue, S.

S. Inoue, Video Microscopy, (Plenum Press, New York1986).

Johansson, J.

S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).

Kondoh, H.

C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
[Crossref]

Lampinen, J.

J. Lampinen and S. Smolander, “Self-organizing feature extraction in recognition of wood surface defects and color images,” Intl. J. Pattern Recognition and Artificial Intelligence,  10, 97–113 (1996).
[Crossref]

Ledbetter, D.H.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Liu, B.

B.-L. Yeo and B. Liu. “Rapid scene analysis on compressed video,” IEEE Trans. Circuits and Systems for Video Technology 5, 533–544 (1995).
[Crossref]

Medioni, G.

P. Ennesser and G. Medioni, “Finding Waldo, or focus of attention using local color information,” IEEE Trans. Pattern Analysis and Machine Intelligence 17, 805–809 (1993).
[Crossref]

Mel, B.

B. Mel, “SEEMORE: Combining color, shape, and texture histogramming in a neurally inspired approach to visual object recognition,” Neural Computation 9, 777–804 (1997).
[Crossref] [PubMed]

Ning, Y.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Palmes-Saloma, C.

C. Palmes-Saloma and C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology 131, 56–66 (2000).
[Crossref]

C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
[Crossref]

Reid, T.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Saloma, C.

C. Palmes-Saloma and C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology 131, 56–66 (2000).
[Crossref]

C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
[Crossref]

M. Soriano and C. Saloma, “Improved classification robustness for noisy cell images represented as principal-component projections in a hybrid recognition system,” Appl. Opt. 37, 3628–3838 (1998).
[Crossref]

Schoell, B.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Schrock, E.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Slater, D.

Smolander, S.

J. Lampinen and S. Smolander, “Self-organizing feature extraction in recognition of wood surface defects and color images,” Intl. J. Pattern Recognition and Artificial Intelligence,  10, 97–113 (1996).
[Crossref]

Soenksen, D.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Soriano, M.

Speicher, M.R.

M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
[Crossref] [PubMed]

Svanderg, S.

S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).

Swain, M.

M. Swain and D. Ballard, “Color indexing,” International J. Computer Vision 7, 11–32 (1991).
[Crossref]

Veldman, T.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Ward, D.

M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
[Crossref] [PubMed]

Weinberg, J.

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Yeo, B.-L.

B.-L. Yeo and B. Liu. “Rapid scene analysis on compressed video,” IEEE Trans. Circuits and Systems for Video Technology 5, 533–544 (1995).
[Crossref]

Appl. Opt. (1)

Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE (1)

S. Andersson-Engels, J. Johansson, and S. Svanderg, “Multicolor fluorescence imaging systems for tissue diagnostics,” Bioimaging Two-Dimensional Spectroscopy, Proc. SPIE 1205, 179–189 (1990).

Biophotonics International (1)

S. Abrams, “Fluorescent Markers: GFP Joins the Common Dyes,” Biophotonics International 5, pp 48–54 (March/April 1998).

IEEE Trans Pattern Analysis Machine Intelligence (1)

B. Funt and G. Finlayson, “Color constant color indexing,” IEEE Trans Pattern Analysis Machine Intelligence 17, 522–529 (1995).
[Crossref]

IEEE Trans. Circuits and Systems for Video Technology (1)

B.-L. Yeo and B. Liu. “Rapid scene analysis on compressed video,” IEEE Trans. Circuits and Systems for Video Technology 5, 533–544 (1995).
[Crossref]

IEEE Trans. Pattern Analysis and Machine Intelligence (1)

P. Ennesser and G. Medioni, “Finding Waldo, or focus of attention using local color information,” IEEE Trans. Pattern Analysis and Machine Intelligence 17, 805–809 (1993).
[Crossref]

International J. Computer Vision (1)

M. Swain and D. Ballard, “Color indexing,” International J. Computer Vision 7, 11–32 (1991).
[Crossref]

Intl. J. Pattern Recognition and Artificial Intelligence (1)

J. Lampinen and S. Smolander, “Self-organizing feature extraction in recognition of wood surface defects and color images,” Intl. J. Pattern Recognition and Artificial Intelligence,  10, 97–113 (1996).
[Crossref]

J. Intelligent Information Systems (1)

C. Faloutsos, W. Equitz, and M. Flickner et al., “Efficient and effective querying by image content,” J. Intelligent Information Systems 3, 231–262 (1994).
[Crossref]

J. Opt. Soc. Am. A (1)

J. Structural Biology (1)

C. Palmes-Saloma and C. Saloma, “Long-depth imaging of specific gene expressions in wholemount mouse embryos with single photon excitation confocal fluorescence microscope and FISH,” J. Structural Biology 131, 56–66 (2000).
[Crossref]

Nature Genetics (1)

M.R. Speicher, S. Gwyn Ballard, and D. Ward, “Karyotyping human chromosomes by combinatorial multifluor FISH,” Nature Genetics 12, 368–375 (1996).
[Crossref] [PubMed]

Neural Computation (1)

B. Mel, “SEEMORE: Combining color, shape, and texture histogramming in a neurally inspired approach to visual object recognition,” Neural Computation 9, 777–804 (1997).
[Crossref] [PubMed]

Phys Med Bio (1)

C. Saloma, C. Palmes-Saloma, and H. Kondoh, “Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium,” Phys Med Bio 43, 1741–1759 (1998).
[Crossref]

Science (1)

E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Weinberg, M.A. Ferguson-Smith, Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, and T. Reid, “Multicolor spectral karyotyping of human chromosomes,” Science 273, 494–498 (1996).
[Crossref] [PubMed]

Other (2)

S. Inoue, Video Microscopy, (Plenum Press, New York1986).

B. Herman, Fluorescence Microscopy2nd Ed. (Springer-Verlag, Singapore1998).

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 (2)

Figure 1.
Figure 1.

Fluorescent microsphere image classes used in recognition experiment. From left to right: R (FITC-red), G (FITC-green), B (AMCA-blue), Bk (background), Gb (FITC-green+AMCA-blue), Rg (FITC-red+FITC-green), Rb (FITC-red+FITC-blue), Rgb (FITC-red+FITC-green+AMCA-blue).

Figure 2.
Figure 2.

Histogram models in rg-space of the eight fluorescent image classes. From left to right, R, G, B, Bk, Gb, Rg, Rb, Rgb. Frequency of pixel occurrence is shown in a rainbow color map with red as the highest and blue the lowest.

Tables (6)

Tables Icon

Table 1. RGB components of the major colors computed using Cluster Mean.

Tables Icon

Table 2. RGB components of the major colors computed using SOFM

Tables Icon

Table 3. Confusion matrix and recognition rates (%RR) for Color Indexing(CI).

Tables Icon

Table 4. Confusion matrix and recognition rates (%RR) for Cluster Mean+Neural Network (CM+NN).

Tables Icon

Table 5. Confusion matrix and recognition rates (%RR) for SOFM+Neural Network (SOFM+NN).

Tables Icon

Table 6. Summary of recognition rates (%RR) for defocused, photobleached and scaled or magnified images.

Equations (3)

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

D = b = 1 B min ( S b , M b ) b = 1 B M b
i ( x ) = arg j min x ( n ) w j
Δ w ( q + 1 ) = k ( dE q / d w ji ) + α Δ w ( q )

Metrics