OSA's Digital Library

Journal of the Optical Society of America A

Journal of the Optical Society of America A


  • Editor: Steven A. Burns
  • Vol. 24, Iss. 9 — Sep. 1, 2007
  • pp: 2622–2627

Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function

Joshua W. Shaevitz and Daniel A. Fletcher  »View Author Affiliations

JOSA A, Vol. 24, Issue 9, pp. 2622-2627 (2007)

View Full Text Article

Enhanced HTML    Acrobat PDF (302 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



We present a technique to systematically measure the change in the blurring function of an optical microscope with distance between the source and the coverglass (the depth) and demonstrate its utility in three-dimensional (3D) deconvolution. By controlling the axial positions of the microscope stage and an optically trapped bead independently, we can record the 3D blurring function at different depths. We find that the peak intensity collected from a single bead decreases with depth and that the width of the axial, but not the lateral, profile increases with depth. We present simple convolution and deconvolution algorithms that use the full depth-varying point-spread functions and use these to demonstrate a reduction of elongation artifacts in a reconstructed image of a 2 μ m sphere.

© 2007 Optical Society of America

OCIS Codes
(100.3020) Image processing : Image reconstruction-restoration
(100.6890) Image processing : Three-dimensional image processing
(110.0180) Imaging systems : Microscopy
(140.7010) Lasers and laser optics : Laser trapping
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Image Processing

Original Manuscript: February 26, 2007
Revised Manuscript: April 23, 2007
Manuscript Accepted: May 1, 2007
Published: July 27, 2007

Virtual Issues
Vol. 2, Iss. 10 Virtual Journal for Biomedical Optics

Joshua W. Shaevitz and Daniel A. Fletcher, "Enhanced three-dimensional deconvolution microscopy using a measured depth-varying point-spread function," J. Opt. Soc. Am. A 24, 2622-2627 (2007)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. D. Agard, Y. Hiraoka, P. Shaw, and J. Sedat, "Fluorescence microscopy in three dimensions," Methods Cell Biol. 30, 353-377 (1989). [CrossRef] [PubMed]
  2. S. Gibson and F. Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 8, 1601-1613 (1991). [CrossRef]
  3. J. Goodman, Introduction to Fourier Optics (Roberts & Co., 2004).
  4. D. Agard, "Optical sectioning microscopy: cellular architecture in three dimensions," Annu. Rev. Biophys. Bioeng. 13, 191-219 (1984). [CrossRef] [PubMed]
  5. B. Scalettar, J. Swedlow, J. Sedat, and D. Agard, "Dispersion, aberration and deconvolution in multi-wavelength fluorescence images," J. Microsc. 182, 50-60 (1996). [CrossRef] [PubMed]
  6. I. Escobar, G. Saavedra, M. Martínez-Corral, and J. Lancis, "Reduction of the spherical aberration effect in high-numerical-aperture optical scanning instruments," J. Opt. Soc. Am. A 23, 3150-3155 (2006). [CrossRef]
  7. J. McNally, C. Preza, J. Conchello, and L. Thomas, Jr., "Artifacts in computational optical-sectioning microscopy," J. Opt. Soc. Am. A 11, 1056-1067 (1994). [CrossRef]
  8. J. Tsujiuchi, "Correction of optical images by compensation of aberrations and by spatial frequency filtering," Prog. Oceanogr. 2, 133-180 (1963).
  9. R. Barakat and A. Houston, "Transfer function of an annular aperture in the presence of spherical aberration," J. Opt. Soc. Am. 55, 538-541 (1965). [CrossRef]
  10. J. Mills and B. Thompson, "Effect of aberrations and apodization on the performance of coherent optical systems. I. The amplitude impulse response," J. Opt. Soc. Am. A 3, 694-703 (1986). [CrossRef]
  11. J. Ojeda-Castafieda, P. Andres, and A. Diaz, "Annular apodizers for low sensitivity to defocus and to spherical aberration," Opt. Lett. 11, 487-489 (1986). [CrossRef]
  12. J. Ojeda-Castaneda, P. Andres, and A. Diaz, "Strehl ratio with low sensitivity to spherical aberration," J. Opt. Soc. Am. A 5, 1233-1236 (1988). [CrossRef]
  13. S. Mezouari and A. Harvey, "Phase pupil functions for reduction of defocus and spherical aberrations," Opt. Lett. 28, 771-773 (2003). [CrossRef] [PubMed]
  14. M. Booth, M. Neil, R. Juskaitis, and T. Wilson, "Adaptive aberration correction in a confocal microscope," Proc. Natl. Acad. Sci. U.S.A. 99, 5788-5792 (2002). [CrossRef] [PubMed]
  15. E. Theofanidou, L. Wilson, W. Hossack, and J. Arlt, "Spherical aberration correction for optical tweezers," Opt. Commun. 236, 145-150 (2004). [CrossRef]
  16. S. Somalingam, K. Dressbach, M. Hain, S. Stankovic, T. Tschudi, J. Knittel, and H. Richter, "Effective spherical aberration compensation by use of a nematic liquid-crystal device," Appl. Opt. 43, 2722-2729 (2004). [CrossRef] [PubMed]
  17. S. Wiersma, P. Török, T. Visser, and P. Varga, "Comparison of different theories for focusing through a plane interface," J. Opt. Soc. Am. A 14, 1482-1490 (1997). [CrossRef]
  18. A. Diaspro, F. Federici, and M. Robello, "Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy," Appl. Opt. 41, 685-690 (2002). [CrossRef] [PubMed]
  19. S. Hell, G. Reiner, C. Cremer, and E. Stelzer, "Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index," J. Microsc. 169, 391-405 (1993). [CrossRef]
  20. N. White, R. Errington, M. Fricker, and J. Wood, "Aberration control in quantitative imaging of botanical specimens by multidimensional fluorescence microscopy," J. Microsc. 181, 99-116 (1996). [CrossRef]
  21. T. Visser, J. Oud, and G. Brakenhoff, "Refractive index and axial distance measurements in 3-d microscopy," Optik (Stuttgart) 90, 17-19 (1992).
  22. K. Neuman, E. Abbondanzieri, and S. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005). [CrossRef] [PubMed]
  23. K. Carlsson, "The influence of specimen refractive index, detector signal integration, and non-uniform scan speed on the imaging properties in confocal microscopy," J. Microsc. 163, 167-178 (1991). [CrossRef]
  24. H. Jacobsen and S. Hell, "Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses," Bioimaging 3, 39-47 (1995). [CrossRef]
  25. T. Visser, G. Brakenhoff, and F. Groen, "The one point response in fluorescence confocal microscopy," Optik (Stuttgart) 87, 39-40 (1991).
  26. T. Visser and J. Oud, "Volume measurements in three-dimensional microscopy," Scanning 16, 198-200 (1994). [CrossRef]
  27. P. Török and P. Varga, "Electromagnetic diffraction of light focused through a stratified medium," Appl. Opt. 36, 2305-2312 (1997). [CrossRef] [PubMed]
  28. C. Sheppard and P. Torok, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997). [CrossRef]
  29. C. Preza and J. Conchello, "Image estimation accounting for point-spread function depth variation in three-dimensional fluorescence microscopy," in Proc. SPIE 4964, 135-142 (2003). [CrossRef]
  30. A. Pralle, E. Florin, E. Stelzer, and J. Hoerber, "Photonic force microscopy: a new tool providing new methods to study membranes at the molecular level," Single Mol. 1, 129-133 (2000). [CrossRef]
  31. K. Svoboda and S. M. Block, "Biological applications of optical forces," Annu. Rev. Biophys. Biomol. Struct. 23, 247-285 (1994). [CrossRef] [PubMed]
  32. E. L. Florin, A. Pralle, J. K. Horber, and E. H. Stelzer, "Photonic force microscope based on optical tweezers and two-photon excitation for biological applications," J. Struct. Biol. 119, 202-211 (1997). [CrossRef] [PubMed]
  33. F. Qian, "Combining optical tweezers and patch clamp for studies of cell membrane electromechanics," Rev. Sci. Instrum. 75, 2937-2942 (2004). [CrossRef] [PubMed]
  34. A. Caspi, R. Granek, and M. Elbaum, "Diffusion and directed motion in cellular transport," Phys. Rev. E 66, 011916 (2002). [CrossRef]
  35. A. Rohrbach and E. H. K. Stelzer, "Trapping forces, force constants, and potential depths for dielectric spheres in the presence of spherical aberrations," Appl. Opt. 41, 2494-2507 (2002). [CrossRef] [PubMed]
  36. http://www.fftw.org/.
  37. M. Lang, C. Asbury, J. Shaevitz, and S. Block, "An automated two-dimensional optical force clamp for single molecule studies," Biophys. J. 83, 491-501 (2002). [CrossRef] [PubMed]
  38. C. Preza and J.-A. Conchello, "Depth-variant maximum-likelihood restoration for three-dimensional fluorescence microscopy." J. Opt. Soc. Am. A 21, 1593-1601 (2004). [CrossRef]
  39. P. Jansson, Deconvolution With Applications in Spectroscopy (Academic, 1984).
  40. P. Crilly, "A quantitative evaluation of various iterative deconvolution algorithms," IEEE Trans. Instrum. Meas. 40, 558-562 (1991). [CrossRef]
  41. A. Boden, D. Redding, R. Hanisch, and J. Mo, "Massively parallel spatially-variant maximum likelihood restoration of Hubble Space Telescope imagery," J. Opt. Soc. Am. A 13, 1537-1545 (1996). [CrossRef]
  42. M. Faisal, A. Lanterman, D. Snyder, and R. White, "Implementation of a modified Richardson-Lucy method for image restoration on a massively parallel computer to compensate for space-variant point spread function of a charge-coupled device camera," J. Opt. Soc. Am. A 12, 2593-2603 (1995). [CrossRef]
  43. J. Markham and J. Conchello, "Fast maximum-likelihood image-restoration algorithms for three-dimensional fluorescence microscopy," J. Opt. Soc. Am. A 18, 1062-1071 (2001). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited