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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 15 — Jul. 29, 2013
  • pp: 17839–17848

Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning

Dong-Ryoung Lee, Young-Duk Kim, Dae-Gab Gweon, and Hongki Yoo  »View Author Affiliations

Optics Express, Vol. 21, Issue 15, pp. 17839-17848 (2013)

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We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

© 2013 OSA

OCIS Codes
(120.3930) Instrumentation, measurement, and metrology : Metrological instrumentation
(180.1790) Microscopy : Confocal microscopy
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy

ToC Category:

Original Manuscript: May 14, 2013
Revised Manuscript: July 12, 2013
Manuscript Accepted: July 14, 2013
Published: July 18, 2013

Virtual Issues
Vol. 8, Iss. 8 Virtual Journal for Biomedical Optics

Dong-Ryoung Lee, Young-Duk Kim, Dae-Gab Gweon, and Hongki Yoo, "Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning," Opt. Express 21, 17839-17848 (2013)

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  1. R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol.21(12), 682–691 (2011). [CrossRef] [PubMed]
  2. E. A. Te Velde, T. Veerman, V. Subramaniam, and T. Ruers, “The use of fluorescent dyes and probes in surgical oncology,” Eur. J. Surg. Oncol.36(1), 6–15 (2010). [CrossRef] [PubMed]
  3. R. Weissleder and V. Ntziachristos, “Shedding light onto live molecular targets,” Nat. Med.9(1), 123–128 (2003). [CrossRef] [PubMed]
  4. D. Li, W. Zheng, and J. Y. Qu, “Two-photon autofluorescence microscopy of multicolor excitation,” Opt. Lett.34(2), 202–204 (2009). [CrossRef] [PubMed]
  5. Y. Hiraoka, J. R. Swedlow, M. R. Paddy, D. A. Agard, and J. W. Sedat, “Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena,” Semin. Cell Biol.2(3), 153–165 (1991). [PubMed]
  6. P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience3(4), 237–242 (2004). [CrossRef] [PubMed]
  7. M. Gu, Principles of Three-dimensional Imaging in Confocal Microscopes (World Scientific, 1996).
  8. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 1995).
  9. R. A. Hoebe, C. H. Van Oven, T. W. Gadella, P. B. Dhonukshe, C. J. Van Noorden, and E. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol.25(2), 249–253 (2007). [CrossRef] [PubMed]
  10. R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J.36(2), 280–290 (2003). [CrossRef] [PubMed]
  11. L. Song, C. A. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy,” Biophys. J.70(6), 2959–2968 (1996). [CrossRef] [PubMed]
  12. J. Huisken and D. Y. Stainier, “Selective plane illumination microscopy techniques in developmental biology,” Development136(12), 1963–1975 (2009). [CrossRef] [PubMed]
  13. T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011). [CrossRef] [PubMed]
  14. S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. Dugast Darzacq, X. Darzacq, C. Wu, C. I. Bargmann, D. A. Agard, M. Dahan, and M. G. Gustafsson, “Fast multicolor 3D imaging using aberration-corrected multifocus microscopy,” Nat. Methods10(1), 60–63 (2012). [CrossRef] [PubMed]
  15. A. K. Ruprecht, T. F. Wiesendanger, and H. J. Tiziani, “Chromatic confocal microscopy with a finite pinhole size,” Opt. Lett.29(18), 2130–2132 (2004). [CrossRef] [PubMed]
  16. H. J. Tiziani and H. M. Uhde, “Three-dimensional image sensing by chromatic confocal microscopy,” Appl. Opt.33(10), 1838–1843 (1994). [CrossRef] [PubMed]
  17. S. Cha, P. C. Lin, L. Zhu, P. C. Sun, and Y. Fainman, “Nontranslational three-dimensional profilometry by chromatic confocal microscopy with dynamically configurable micromirror scanning,” Appl. Opt.39(16), 2605–2613 (2000). [CrossRef] [PubMed]
  18. T. Kim, S. H. Kim, D. Do, H. Yoo, and D. Gweon, “Chromatic confocal microscopy with a novel wavelength detection method using transmittance,” Opt. Express21(5), 6286–6294 (2013). [CrossRef] [PubMed]
  19. K. B. Shi, P. Li, S. Z. Yin, and Z. W. Liu, “Chromatic confocal microscopy using supercontinuum light,” Opt. Express12(10), 2096–2101 (2004). [CrossRef] [PubMed]
  20. Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two-photon excitation fluorescence imaging,” J. Microsc.235(1), 79–83 (2009). [CrossRef] [PubMed]
  21. C. A. Yang, K. B. Shi, H. F. Li, Q. A. Xu, V. Gopalan, and Z. W. Liu, “Chromatic second harmonic imaging,” Opt. Express18(23), 23837–23843 (2010). [CrossRef] [PubMed]
  22. C. J. R. Sheppard and C. J. Cogswell, “Confocal microscopy with detector arrays,” J. Mod. Opt.37(2), 267–279 (1990). [CrossRef]
  23. C. H. Lee and J. P. Wang, “Noninterferometric differential confocal microscopy with 2-nm depth resolution,” Opt. Commun.135(4-6), 233–237 (1997). [CrossRef]
  24. W. Q. Zhao, J. B. Tan, and L. R. Qiu, “Bipolar absolute differential confocal approach to higher spatial resolution,” Opt. Express12(21), 5013–5021 (2004). [CrossRef] [PubMed]
  25. J. B. Tan, J. Liu, and Y. H. Wang, “Differential confocal microscopy with a wide measuring range based on polychromatic illumination,” Meas. Sci. Technol.21(5), 054013 (2010). [CrossRef]
  26. Y. Wang, L. R. Qiu, Y. X. Song, and W. Q. Zhao, “Laser differential confocal lens thickness measurement,” Meas. Sci. Technol.23(5), 055204 (2012). [CrossRef]
  27. W. Q. Zhao, C. Liu, and L. R. Qiu, “Laser divided-aperture differential confocal sensing technology with improved axial resolution,” Opt. Express20(23), 25979–25989 (2012). [CrossRef] [PubMed]
  28. L. M. Zou, J. Q. Qu, S. L. Hou, and X. M. Ding, “Differential confocal technology based on radial birefringent pupil filtering principle,” Opt. Commun.285(8), 2022–2027 (2012). [CrossRef]
  29. A. Bilenca, A. Ozcan, B. Bouma, and G. Tearney, “Fluorescence coherence tomography,” Opt. Express14(16), 7134–7143 (2006). [CrossRef] [PubMed]
  30. M. de Groot, C. L. Evans, and J. F. de Boer, “Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning,” Opt. Express20(14), 15253–15262 (2012). [CrossRef] [PubMed]
  31. T. Wilson and A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett.12(4), 227–229 (1987). [CrossRef] [PubMed]
  32. M. Gu and C. J. R. Sheppard, “Confocal fluorescent microscopy with a finite-sized circular detector,” J. Opt. Soc. Am. A9(1), 151–153 (1992). [CrossRef]
  33. J. W. Goodman, Introduction to Fourier Optics (Roberts & Company, 2005).

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