3D Deconvolution Microscopy

David S.C. Biggs1

1 KB Imaging Solutions LLC, Waterford, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.19
DOI:  10.1002/0471142956.cy1219s52
Online Posting Date:  April, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


3D deconvolution microscopy is a combination of optical and computational techniques that are used to maximize the observed resolution and signal from a biological specimen. Mathematical models are used to predict the distribution of out‐of‐focus light caused by the inherent optical limitations of the instrument, which can then be compensated for using computer algorithms. This unit will review the theory of image formation and characteristics of the point spread function (PSF) based on the instrument modality and objective lens parameters. A variety of commonly used deblurring and deconvolution methods are described, and their applications to sample datasets are illustrated to show the performance of each algorithm. Steps for setting up the image acquisition to acquire data suitable for deconvolution are described, and the challenge of maximizing signal levels while minimizing light exposure addressed. Deconvolution examples from widefield epi‐fluorescence and laser scanning confocal are shown, and suitability for other modalities discussed. Curr. Protoc. Cytom. 52:12.19.1‐12.19.20. © 2010 by John Wiley & Sons, Inc.

Keywords: deconvolution; deblurring; image restoration; point spread function; fluorescence; widefield; confocal

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Image Formation
  • Resolution and Sampling
  • Estimating and Optimizing the PSF
  • Deblurring and Deconvolution Algorithms
  • Blind Deconvolution
  • Example Deconvolution Results
  • Deconvolution Software
  • Basic Protocol 1: Data Acquisition and Deconvolution Analysis
  • Concluding Remarks
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Data Acquisition and Deconvolution Analysis

  • Research‐grade widefield optical microscope (upright or inverted)
  • Monochrome CCD camera, sensitive to low light fluorescence, cooled, 6 to 7 µm pixels, 12‐ to16‐bit images, sufficient sensor size to cover the desired field of view
  • Plan apochromat/plan fluorite objective lenses (with correction collar if available)
  • Motorized Z‐stage or piezo‐electric focusing mechanism with nanometer resolution and repeatability
  • Uniform fluorescence slide to check illumination uniformity
  • Stage micrometer to calibrate the pixel spacing from the sample to the camera
  • Light source with stable emission intensity
  • Computer‐controlled excitation shutter
  • Filter sets matched to the excitation and emission of the fluorescence involved
  • Brightfield, phase, or DIC optics for initial specimen observation
  • Acquisition software to control the microscope, Z focusing, camera setup and capture, shutter operation, live preview, automated image capture, and file saving
  • Multi‐spectral sub‐resolution fluorescence beads, to check the lateral and axial alignment between multiple wavelengths, and to identify any unwanted aberrations or nonsymmetries in the PSF
NOTE: By their nature, laser scanning confocal instruments are normally already an integrated hardware and software solution suitable for automated 3D acquisition.
PDF or HTML at Wiley Online Library



Literature Cited

   Agard, D.A. 1984. Optical sectioning microscopy: Cellular architecture in three dimensions. Annu. Rev. Biophys. Bioeng. 13:191‐219.
   Arnison, M.R. 2004. Phase Control and Measurement in Digital Microscopy. Ph.D. Thesis, University of Sydney, Australia.
   Biggs, D.S.C. 1998. Accelerated Iterative Blind Deconvolution. Ph.D. Thesis, University of Auckland, New Zealand. http://researchspace.auckland.ac.nz/handle/2292/1760.
   Biggs, D.S.C. 2010. A practical guide to deconvolution of fluorescence microscope imagery. Microscopy Today 18, No. 1.
   Born, M. and Wolf, E. 1999. Principles of Optics, 7th edition. Cambridge University Press. Cambridge.
   Cannell, M.B., McMorland, A., and Soeller, C. 2006. Image enhancement by deconvolution. In Handbook of Biological Confocal Microscopy, 3rd ed. (J. Pawley, ed.) pp. 488‐500. Springer, New York.
   Fish, D.A., Brinicombe, A.M., Pike, E.R., and Walker, J.G. 1995. Blind deconvolution by means of the Richardson‐Lucy algorithm. J. Opt. Soc. Am. A 12:58‐65.
   Gold, R. 1964. An Iterative Unfolding Method for Matrices, Tech. Rep. ANL‐6984. Argonne National Laboratory, Argonne, Illinois.
   Gonzalez, R.C. and Woods, R.E. 2007. Digital Image Processing, Prentice Hall, Upper Saddle River, N.J.
   Holmes, T.J., Biggs, D., and Abu‐Tarif, A. 2006. Blind deconvolution. In Handbook of Biological Confocal Microscopy, 3rd ed. (J. Pawley, ed.) pp. 468‐487. Springer, New York.
   Larson, J. 2002. Two‐dimensional and three‐dimensional blind deconvolution of fluorescence confocal images. Proc. SPIE 86:4621.
   Lucy, L.B. 1974. An iterative technique for the rectification of observed distributions. Astron. J. 79:745‐754.
   Richardson, W.H. 1972. Bayesian‐based iterative method of image restoration. J. Opt. Soc. Am. 62:55‐59.
   Sibarita, J.B. 2005. Deconvolution microscopy. Adv. Biochem. Engin./Biotechnol. 95:201‐243.
Key References
   Biggs, D.S.C. 2004. Clearing up deconvolution. Biophotonics Int. February:32‐37.
  For short overview articles on deconvolution and microscopy:
   Wallace, W., Schaefer, L.H., and Swedlow, J.R. 2001. Working person's guide to deconvolution in light microscopy. BioTechniques 31:1076‐1097.
  For a more in‐depth review of deconvolution techniques:
   Sibarita, 2005. See above.
  For a more technical review of deconvolution algorithms:
   Holmes, et al., 2006. See above.
  For a comprehensive reference book on biological confocal (and widefield) microscopy:
   Sarder, P. and Nehorai, A. 2006. Deconvolution methods for 3D fluorescence microscopy images: An overview. IEEE Signal Proc. Mag. 23:32‐45.
   Pawley, J.B. (ed.) 2006. Handbook of Biological Confocal Microscopy, 3rd ed. Springer, New York.
Internet Resources
  An easily accessible reference to microscopy principles, usage and applications can be found at the Molecular Expressions Web site.
  Deconvolution section at Molecular Expressions.
  A tutorial Web site for 3D deconvolution that extends the work presented in this unit, and includes more example results.
PDF or HTML at Wiley Online Library