Abstract

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

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2008 (3)

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]

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]

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]

2007 (6)

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]

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]

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]

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]

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]

J. X. Cheng, “Coherent anti-Stokes Raman Scattering microscopy,” Appl. Spectrosc. 61, 197A-208A (2007).
[CrossRef]

2006 (8)

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]

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]

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]

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]

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]

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]

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]

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]

2005 (3)

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]

G. Cox, N. Moreno, and J. Feijo, “Second-harmonic imaging of plant polysaccharides,” J. Biomed. Opt. 10, 024013 (2005).
[CrossRef] [PubMed]

V. Barzda, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13, 8263-8276 (2005).
[CrossRef] [PubMed]

2004 (4)

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]

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]

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]

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]

2003 (6)

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]

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]

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]

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]

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]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1368-1376 (2003).
[CrossRef]

2002 (1)

2000 (2)

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).

L. Moreaux, O. Sandre, and J. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685-1694 (2000).
[CrossRef]

1999 (1)

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]

1996 (1)

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]

1991 (1)

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]

1985 (1)

R. M. Haralick and L. G. Shapiro, “Image segmentation techniques,” Comput. Vis. Graph. Image Process. 29, 100-132 (1985).
[CrossRef]

1978 (1)

N. Otsu, “A threshold selection method from gray-level histogram,” IEEE Trans. Syst. Man Cybern. 8, 62-66 (1978).

Abdul-Karim, M.-A.

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]

Akimov, D.

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]

Alfano, R. R.

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]

Alfimov, M. V.

Al-Kofahi, K.

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]

Al-Kofahi, O.

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]

Axäng, C.

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]

Barzda, V.

Beaurepaire, E.

Belisle, J. M.

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]

Bellemare, M. J.

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]

Billard, F.

Bohle, D. S.

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]

Boucher, Y.

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]

Boulesteix, T.

Brackmann, C.

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]

Brasselet, S.

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]

Brown, E.

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]

Brown, R. M.

Campagnola, P. J.

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]

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]

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]

Chatzipapadopoulos, S.

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]

Chen, J. S.

Chen, S. Y.

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]

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]

Chen, Y. C.

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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]

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]

Simpkins, B. S.

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]

Soille, P.

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]

Sulston, J.

J. Sulston and J. Hodgkin, “Methods,” in The Nematode Caenorhabditis elegans, W.B.Wood, ed. (Cold Spring Harbor Laboratory Press, 1988), pp. 587-606.

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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]

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[CrossRef]

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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).
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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).
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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).
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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]

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[CrossRef] [PubMed]

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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).
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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]

Tsai, T. F.

Tsai, T. H.

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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.
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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).
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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]

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).
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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).
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W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1368-1376 (2003).
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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).
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R. C. Gonzales and R. E. Woods, “Thresholding,” in Digital Image Processing (Prentice-Hall, 2002), pp. 595-611.

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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).
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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).
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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).
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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).
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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).
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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]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1368-1376 (2003).
[CrossRef]

Zoumi, A.

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]

Zyss, J.

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).
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Appl. Phys. Lett. (2)

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).
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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]

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]

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).
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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]

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]

Chem. Phys. Lett. (1)

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).
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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).
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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]

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]

Nano Lett. (1)

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).
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Nat. Biotechnol. (2)

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).
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W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1368-1376 (2003).
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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).
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Nat. Methods (1)

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]

Opt. Express (3)

Opt. Lett. (5)

Proc. Natl. Acad. Sci. U.S.A. (3)

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).
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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).
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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]

J. Sulston and J. Hodgkin, “Methods,” in The Nematode Caenorhabditis elegans, W.B.Wood, ed. (Cold Spring Harbor Laboratory Press, 1988), pp. 587-606.

R. C. Gonzales and R. E. Woods, “Thresholding,” in Digital Image Processing (Prentice-Hall, 2002), pp. 595-611.

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]

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Figures (7)

Fig. 1
Fig. 1

Optical process involving the interaction of the pump, Stokes, and probe breams for the generation of a CARS signal is schematically illustrated in the energy level diagram in (a), where Ω represents the molecular vibration. Below, the experimental setup used for the generation of CARS microscopy images of polystyrene spheres, living yeast cells, and nematodes is shown. The probe geometry assumed in the theoretical model is depicted in (b), showing the focused excitation beams scanning the object (a polystyrene sphere), as well as the coordinate system of a detection point in the far-field regime.

Fig. 2
Fig. 2

Experimentally generated CARS microscopy images of polystyrene spheres of sizes 1.072 μ m and 2.836 μ m are shown in (a) and (b). In (c) and (d) the profiles along the lines indicated in the images are compared with profiles sampled from theoretically generated images of corresponding spheres. Except for the borders of interference, an excellent match ( 0.9 % and 2 % , respectively) is obtained, which validates the use of the theoretical model for systematic evaluation of image analysis routines within CARS microscopy.

Fig. 3
Fig. 3

Series of theoretically generated images of polystyrene spheres with diameters of 0.5, 1, and 2 μ m are shown in (a). The dashed circles highlight the measure of FWHM. The corresponding normalized profiles are shown in the diagrams below with the FWHM marked with dashed vertical lines. Compared to the true size, indicated by cross marks, the FWHM provides a significantly smaller diameter.

Fig. 4
Fig. 4

Relative error in the size estimates of polystyrene spheres in the size range of 0.5 2 μ m obtained from the theoretical images using different image analysis algorithms are plotted versus the true diameters. Whereas local thresholding and level sets identify object sizes with a high accuracy in the range of D = 0.7 2 μ m , global thresholding and watersheds perform almost as poorly as the FWHM parameter.

Fig. 5
Fig. 5

CARS microscopy images of polystyrene spheres with sizes 1.072 μ m and 2.836 μ m are shown in (a) and (b) respectively. The white circles mark the area identified as object by the level sets algorithm. The corresponding intensity profiles are displayed in (c) and (d), also showing the borders as defined by level sets (circles) and the FWHM (vertical lines). For comparison, the true sizes of the spheres are indicated by cross marks.

Fig. 6
Fig. 6

CARS microscopy image of lipid droplets in living yeast cells is shown in (a). A close-up of the rectangular area highlighted in (a) is displayed in (b). The borders of the lipid droplet as identified by global thresholding, watersheds, local thresholding, and level sets are shown in (c), (d), (e), and (f), respectively. Whereas watersheds and local thresholding are successful in identifying the lipid droplet, global thresholding and level sets rather identify the surrounding structure corresponding to the outline of the entire cell.

Fig. 7
Fig. 7

CARS microscopy images of lipid stores in living C. elegans were collected in the region indicated in the schematic illustration of a nematode in (a). An example of such an image is shown in (b), clearly visualizing the lipid stores as adjacent circular objects. The borders of the lipid stores as identified by global thresholding, watersheds, local thresholding, and level sets are shown in (c), (d), (e), and (f), respectively. It can be noted that global thresholding (c) tends to identify the lipid stores as larger clusters, being unable to separate the single droplets. Whereas level sets (f) fails to identify many of the lipid stores, local thresholding (e) provides a series of false positives at the right edge of the image. Combined with an initial blob filtering of the image, watersheds (d) is able to successfully identify most of the important lipid stores as single objects without any accompanying false positives.

Tables (3)

Tables Icon

Table 1 Segmentation of Theoretical CARS Microscopy Images of Polystyrene Spheres With and Without the Border of Destructive Interference a

Tables Icon

Table 2 Segmentation of Experimentally Generated CARS Microscopy Images of Polystyrene Spheres with Two Different Diameters a

Tables Icon

Table 3 Segmentation of CARS Microscopy Images of Lipid Droplets in Living Yeast Cells Showing the Impact of a Structured Surrounding a

Equations (11)

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

I CARS χ ( 3 ) 2 I p I S I p ,
χ ( 3 ) = χ r ( 3 ) + χ n r ( 3 ) ,
E p ( r , t ) = E p ( r ) exp ( i ω p t ) + c.c. ,
E S ( r , t ) = E S ( r ) exp ( i ω S t ) + c.c.
E j inc ( α ) = E j 0 exp ( f 2 sin 2 α ω 0 2 ) ,
E j ( ρ , ϕ , z ) = i k j f exp ( i k j f ) 2 I 00
I 00 = 0 α max E j inc ( α ) sin α cos α ( 1 + cos α ) J 0 ( k j ρ sin α ) exp ( i k j x cos α ) ,
E aS ( R ) = ω aS 2 c 2 exp ( i k aS R ) R V d V exp ( i k aS R r R ) × [ 0 0 0 cos ϴ cos Φ cos ϴ sin Φ sin ϴ sin Φ cos Φ 0 ] × [ P x ( 3 ) ( r ) P y ( 3 ) ( r ) P z ( 3 ) ( r ) ] i ̂ R i ̂ ϴ i ̂ Φ ,
P x ( 3 ) ( r ) = 3 χ 1111 ( 3 ) ( ω aS , r ) E p 2 ( r ) E S * ( r ) ,
p CARS = n as c 8 π ϴ 1 ϴ 2 d ϴ 0 2 π d Φ E aS ( R ) 2 R 2 sin ϴ .
F ( c 1 , c 2 , C ) = μ Length ( C ) + ν Area ( inside ( C ) ) + λ 1 inside ( C ) u 0 ( x , y ) c 1 2 d x d y + λ 2 outside ( C ) u 0 ( x , y ) c 2 2 d x d y ,

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