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Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Editor: Stephen A. Burns
  • Vol. 25, Iss. 9 — Sep. 1, 2008
  • pp: 2195–2206

Image analysis in nonlinear microscopy

Jonas Hagmar, Christian Brackmann, Tomas Gustavsson, and Annika Enejder  »View Author Affiliations

JOSA A, Vol. 25, Issue 9, pp. 2195-2206 (2008)

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The ability to automatically extract quantitative data from nonlinear microscopy images is here explored, taking nonlinear and coherent effects into account. Objects of different degrees of complexity were investigated: theoretical images of spherical objects, experimentally collected coherent anti-Stokes Raman scattering images of polystyrene spheres in background-generating agar, well-separated lipid droplets in living yeast cells, and conglomerations of lipid droplets in living C. elegans nematodes. The in linear microscopy useful measure of full width at half-maximum (FWHM) was shown to provide inadequate measures of object size due to the nonlinear density dependence of the signal. Instead, the capability of four state-of-the-art image analysis algorithms was evaluated. Among these, local thresholding was found to be the widest applicable segmentation algorithm.

© 2008 Optical Society of America

OCIS Codes
(100.2960) Image processing : Image analysis
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: March 17, 2008
Revised Manuscript: June 21, 2008
Manuscript Accepted: June 24, 2008
Published: August 6, 2008

Virtual Issues
Vol. 3, Iss. 11 Virtual Journal for Biomedical Optics

Jonas Hagmar, Christian Brackmann, Tomas Gustavsson, and Annika Enejder, "Image analysis in nonlinear microscopy," J. Opt. Soc. Am. A 25, 2195-2206 (2008)

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  1. P. J. Shaw, “Comparison of widefield/deconvolution microscopy for three dimensional imaging,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.Pawley, ed. (Springer, 2006), pp. 453-457. [CrossRef]
  2. R. M. Haralick and L. G. Shapiro, “Image segmentation techniques,” Comput. Vis. Graph. Image Process. 29, 100-132 (1985). [CrossRef]
  3. S. C. Zhu and A. Yuille, “Region competition: unifying snakes, region growing, and Bayes/MDL for multiband image segmentation,” IEEE Trans. Pattern Anal. Mach. Intell. 18, 884-900 (1996). [CrossRef]
  4. B. Roysam, G. Lin, M.-A. Abdul-Karim, O. Al-Kofahi, K. Al-Kofahi, W. Shain, D. H. Szarowsk, and J. N. Turner, “Automated three dimensional image analysis methods for confocal microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.Pawley, ed. (Springer, 2006), pp. 316-337. [CrossRef]
  5. L. Moreaux, O. Sandre, and J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685-1694 (2000). [CrossRef]
  6. L. Li and J. X. Cheng, “Label-free coherent anti-Stokes Raman scattering imaging of coexisting lipid domains in single bilayers,” J. Phys. Chem. B 112, 1576-1579 (2008). [CrossRef] [PubMed]
  7. X. L. Nan, E. O. Potma, and X. S. Xie, “Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-Stokes Raman scattering microscopy,” Biophys. J. 91, 728-735 (2006). [CrossRef] [PubMed]
  8. D. Debarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3, 47-53 (2006). [CrossRef]
  9. T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, “Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy,” Proc. Natl. Acad. Sci. U.S.A. 104, 14658-14663 (2007). [CrossRef] [PubMed]
  10. Y. C. Guo, H. E. Savage, F. Liu, S. P. Schantz, P. P. Ho, and R. R. Alfano, “Subsurface tumor progression investigated by noninvasive optical second harmonic tomography,” Proc. Natl. Acad. Sci. U.S.A. 96, 10854-10856 (1999). [CrossRef] [PubMed]
  11. E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. (N.Y.) 9, 796-800 (2003). [CrossRef]
  12. S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, “Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging,” Opt. Lett. 31, 2756-2758 (2006). [CrossRef] [PubMed]
  13. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356-1360 (2003). [CrossRef] [PubMed]
  14. T. Boulesteix, E. Beaurepaire, M. P. Sauviat, and M. C. Schanne-Klein, “Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy,” Opt. Lett. 29, 2031-2033 (2004). [CrossRef] [PubMed]
  15. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90, 693-703 (2006). [CrossRef]
  16. A. Zoumi, X. A. Lu, G. S. Kassab, and B. J. Tromberg, “Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy,” Biophys. J. 87, 2778-2786 (2004). [CrossRef] [PubMed]
  17. V. Barzda, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13, 8263-8276 (2005). [CrossRef] [PubMed]
  18. J. M. Belisle, S. Costantino, M. L. Leimanis, M. J. Bellemare, D. S. Bohle, E. Georges, and P. W. Wiseman, “Sensitive detection of malaria infection by third harmonic generation imaging,” Biophys. J. 94, L26-L28 (2008). [CrossRef]
  19. D. A. Dombeck, K. A. Kasischke, H. D. Vishwasrao, M. Ingelsson, B. T. Hyman, and W. W. Webb, “Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy,” Proc. Natl. Acad. Sci. U.S.A. 100, 7081-7086 (2003). [CrossRef] [PubMed]
  20. H. F. Wang, Y. Fu, P. Zickmund, R. Y. Shi, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues,” Biophys. J. 89, 581-591 (2005). [CrossRef] [PubMed]
  21. Y. Fu, H. F. Wang, R. Y. Shi, and J. X. Cheng, “Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues,” Biophys. J. 92, 3251-3259 (2007). [CrossRef] [PubMed]
  22. S. W. Chu, S. Y. Chen, T. H. Tsai, T. M. Liu, C. Y. Lin, H. J. Tsai, and C. K. Sun, “In vivo developmental biology study using noninvasive multi-harmonic generation microscopy,” Opt. Express 11, 3093-3099 (2003). [CrossRef] [PubMed]
  23. D. Debarre, W. Supatto, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 2881-2883 (2004). [CrossRef]
  24. S. Y. Chen, C. S. Hsieh, S. W. Chu, C. Y. Lin, C. Y. Ko, Y. C. Chen, H. J. Tsai, C. H. Hu, and C. K. Sun, “Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo,” J. Biomed. Opt. 11, 054022 (2006). [CrossRef] [PubMed]
  25. R. M. Brown, A. C. Millard, and P. J. Campagnola, “Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy,” Opt. Lett. 28, 2207-2209 (2003). [CrossRef] [PubMed]
  26. G. Cox, N. Moreno, and J. Feijo, “Second-harmonic imaging of plant polysaccharides,” J. Biomed. Opt. 10, 024013 (2005). [CrossRef] [PubMed]
  27. T. Meyer, D. Akimov, N. Tarcea, S. Chatzipapadopoulos, G. Muschiolik, J. Kobow, M. Schmitt, and J. Popp, “Three-dimensional molecular mapping of a multiple emulsion by means of CARS microscopy,” J. Phys. Chem. B 112, 1420-1426 (2008). [CrossRef] [PubMed]
  28. E. O. Potma, X. S. Xie, L. Muntean, J. Preusser, D. Jones, J. Ye, S. R. Leone, W. D. Hinsberg, and W. Schade, “Chemical imaging of photoresists with coherent anti-Stokes Raman scattering (CARS) microscopy,” J. Phys. Chem. B 108, 1296-1301 (2004). [CrossRef]
  29. T. Manaka, E. Lim, R. Tamura, D. Yamada, and M. Iwamoto, “Probing of the electric field distribution in organic field effect transistor channel by microscopic second-harmonic generation,” Appl. Phys. Lett. 89, 072113 (2006). [CrossRef]
  30. V. P. Mitrokhin, A. B. Fedotov, A. A. Ivanov, M. V. Alfimov, and A. M. Zheltikov, “Coherent anti-Stokes Raman scattering microspectroscopy of silicon components with a photonic-crystal fiber frequency shifter,” Opt. Lett. 32, 3471-3473 (2007). [CrossRef] [PubMed]
  31. E. Delahaye, N. Tancrez, T. Yi, I. Ledoux, J. Zyss, S. Brasselet, and R. Clement, “Second harmonic generation from individual hybrid MnPS3-based nanoparticles investigated by nonlinear microscopy,” Chem. Phys. Lett. 429, 533-537 (2006). [CrossRef]
  32. J. P. Long, B. S. Simpkins, D. J. Rowenhorst, and P. E. Pehrsson, “Far-field imaging of optical second-harmonic generation in single GaN nanowires,” Nano Lett. 7, 831-836 (2007). [CrossRef] [PubMed]
  33. J.-X. Cheng, A. Volkmer, and X. S. Xie, “Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy,” J. Opt. Soc. Am. B 19, 1363-1375 (2002). [CrossRef]
  34. I. D. Nikolov and C. D. Ivanov, “Optical plastic refractive measurements in the visible and the near-infrared regions,” Appl. Phys. Lett. 39, 2067-2070 (2000).
  35. D. Gachet, F. Billard, N. Sandeau, and H. Rigneault, “Coherent anti-Stokes Raman scattering (CARS) microscopy imaging at interfaces: evidence of interference effects,” Opt. Express 15, 10408-10420 (2007). [CrossRef] [PubMed]
  36. J. Sulston and J. Hodgkin, “Methods,” in The Nematode Caenorhabditis elegans, W.B.Wood, ed. (Cold Spring Harbor Laboratory Press, 1988), pp. 587-606.
  37. R. C. Gonzales and R. E. Woods, “Thresholding,” in Digital Image Processing (Prentice-Hall, 2002), pp. 595-611.
  38. N. Otsu, “A threshold selection method from gray-level histogram,” IEEE Trans. Syst. Man Cybern. 8, 62-66 (1978).
  39. L. Vincent and P. Soille, “Watersheds in digital spaces: an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 13, 583-598 (1991). [CrossRef]
  40. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1368-1376 (2003). [CrossRef]
  41. M. Oheim, D. J. Michael, M. Geisbauer, D. Madsen, and R. H. Chow, “Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches,” Adv. Drug Delivery Rev. 58, 788-808 (2006). [CrossRef]
  42. J. X. Cheng, “Coherent anti-Stokes Raman Scattering microscopy,” Appl. Spectrosc. 61, 197A-208A (2007). [CrossRef]

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