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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 21 — Oct. 11, 2010
  • pp: 22324–22338

Removal of subsurface fluorescence in cryo-imaging using deconvolution

Ganapathy Krishnamurthi, Charlie Y. Wang, Grant Steyer, and David L. Wilson  »View Author Affiliations

Optics Express, Vol. 18, Issue 21, pp. 22324-22338 (2010)

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We compared image restoration methods [Richardson-Lucy (RL), Wiener, and Next-image] with measured “scatter” point-spread-functions, for removing subsurface fluorescence from section-and-image cryo-image volumes. All methods removed haze, delineated single cells from clusters, and improved visualization, but RL best represented structures. Contrast-to-noise and contrast-to-background improvement from RL and Wiener were comparable and 35% better than Next-image. Concerning detection of labeled cells, ROC analyses showed RL ≈Wiener > Next-image >> no processing. Next-image was faster than other methods and less prone to image processing artifacts. RL is recommended for the best restoration of the shape and size of fluorescent structures.

© 2010 OSA

OCIS Codes
(100.0100) Image processing : Image processing
(100.1830) Image processing : Deconvolution
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.0180) Microscopy : Microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Image Processing

Original Manuscript: August 6, 2010
Revised Manuscript: September 12, 2010
Manuscript Accepted: September 20, 2010
Published: October 7, 2010

Virtual Issues
Vol. 5, Iss. 14 Virtual Journal for Biomedical Optics

Ganapathy Krishnamurthi, Charlie Y. Wang, Grant Steyer, and David L. Wilson, "Removal of subsurface fluorescence in cryo-imaging using deconvolution," Opt. Express 18, 22324-22338 (2010)

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  1. G. J. Steyer, D. Roy, O. Salvado, M. E. Stone, and D. L. Wilson, “Cryo-Imaging of Fluorescently-Labeled Single Cells in a Mouse,” Proc. Soc. Photo Opt. Instrum. Eng. 7262, 72620W, W8 (2009). [PubMed]
  2. D. Roy, G. J. Steyer, M. Gargesha, M. E. Stone, and D. L. Wilson, “3D cryo-imaging: a very high-resolution view of the whole mouse,” Anat. Rec. (Hoboken) 292(3), 342–351 (2009). [CrossRef]
  3. M. Gargesha, M. Qutaish, D. Roy, G. Steyer, H. Bartsch, and D. L. Wilson, “Enhanced Volume Rendering Techniques for High-Resolution Color Cryo-Imaging Data,” Proc. Soc. Photo Opt. Instrum. Eng. 7262, 72655V (2009). [PubMed]
  4. G. J. Steyer, D. Roy, O. Salvado, M. E. Stone, and D. L. Wilson, “Removal of out-of-plane fluorescence for single cell visualization and quantification in cryo-imaging,” Ann. Biomed. Eng. 37(8), 1613–1628 (2009). [CrossRef] [PubMed]
  5. D. Roy, M. Gargesha, G. J. Steyer, P. Hakimi, R. W. Hanson, and D. L. Wilson, “Multi-scale Characterization of the PEPCK-Cmus Mouse Through 3D Cryo-Imaging,” Int. J. Biomed. Imaging 2010, 1–12 (2010). [CrossRef]
  6. J. A. Spaan, R. ter Wee, J. W. van Teeffelen, G. Streekstra, M. Siebes, C. Kolyva, H. Vink, D. S. Fokkema, and E. VanBavel, “Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas,” Med. Biol. Eng. Comput. 43(4), 431–435 (2005). [CrossRef] [PubMed]
  7. P. S. Tsai, B. Friedman, A. I. Ifarraguerri, B. D. Thompson, V. Lev-Ram, C. B. Schaffer, Q. Xiong, R. Y. Tsien, J. A. Squier, and D. Kleinfeld, “All-optical histology using ultrashort laser pulses,” Neuron 39(1), 27–41 (2003). [CrossRef] [PubMed]
  8. T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. So, “High-resolution whole organ imaging using two-photon tissue cytometry,” J. Biomed. Opt. 12(1), 014015 (2007). [CrossRef] [PubMed]
  9. J. Mitić, T. Anhut, M. Meier, M. Ducros, A. Serov, and T. Lasser, “Optical sectioning in wide-field microscopy obtained by dynamic structured light illumination and detection based on a smart pixel detector array,” Opt. Lett. 28(9), 698–700 (2003). [CrossRef] [PubMed]
  10. M. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef]
  11. G. D. Li, D. Savvas, E. Arthur, and E. J. Lucy, “A new wide field-of-view confocal imaging system and its applications in drug discovery and pathology,” Proc. Soc. Photo Opt. Instrum. Eng. 6009, 1–16 (2005).
  12. P. van Horssen, M. Siebes, I. Hoefer, J. A. Spaan, and J. P. van den Wijngaard, “Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome,” Med. Biol. Eng. Comput. 48(8), 735–744 (2010). [CrossRef] [PubMed]
  13. P. Sarder and A. Nehorai, “Deconvolution methods for 3-D fluorescence microscopy images,” IEEE Signal Process. Mag. 23(3), 32–45 (2006). [CrossRef]
  14. W. Wallace, L. H. Schaefer, and J. R. Swedlow, “A workingperson’s guide to deconvolution in light microscopy,” Biotechniques 31(5), 1076–1078, 1080, 1082 passim (2001). [PubMed]
  15. L. B. Lucy, “An iterative technique for the rectification of observed distributions,” Astron. J. 79, 745–754 (1974). [CrossRef]
  16. W. H. Richardson, “Bayesian-Based Iterative Method of Image Restoration,” J. Opt. Soc. Am. 62(1), 55–59 (1972). [CrossRef]
  17. N. Wiener, ed., Extrapolation, Interpolation, and Smoothing of Stationary Time Series (Wiley, New York, 1949).
  18. L. A. Shepp and Y. Vardi, “Maximum likelihood reconstruction for emission tomography,” IEEE Trans. Med. Imaging 1(2), 113–122 (1982). [CrossRef] [PubMed]
  19. R. L. White, “Image restoration using the damped Richardson-Lucy method,” Proc. Soc. Photo Opt. Instrum. Eng. 2198, 1342–1348 (1994).
  20. J. C. Szucsik, A. G. Lewis, D. J. Marmer, and J. L. Lessard, “Urogenital tract expression of enhanced green fluorescent protein in transgenic mice driven by a smooth muscle gamma-actin promoter,” J. Urol. 171(2), 944–949 (2004). [CrossRef] [PubMed]
  21. S. H. Park, J. M. Goo, and C. H. Jo, “Receiver operating characteristic (ROC) curve: practical review for radiologists,” Korean J. Radiol. 5(1), 11–18 (2004). [CrossRef] [PubMed]

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