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Microscopy Reference Library

Deconvolution Microscopy

Over the past several decades, deconvolution microscopy has become a mainstream image processing tool for deciphering the substructure of living and fixed specimens in three dimensions. Routinely applied to widefield optical sections, as well as those obtained in confocal and structured illumination, the technique has benefited from the continued development of advanced algorithms and turnkey systems. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of deconvolution.

Sibarita, J. B.

Deconvolution microscopy.  Advances in Biochemical Engineering/Biotechnology 95: 201-243 (2005).  The author presents a comprehensive review of deconvolution methodology in widefield fluorescence microscopy. Included are a review of optical sectioning, distance calibration, optical aberrations, the point spread function, detectors, sampling, microscope configuration details, and image acquisition. In addition, the most useful deconvolution algorithms are described in detail.

Swedlow, J. R. and Platani, M.

Live cell imaging using wide-field microscopy and deconvolution.  Cell Structure and Function 27: 335-341 (2002).  An excellent review on the application of deconvolution fluorescence microscopy to live-cell imaging using fluorescent proteins. The authors discuss a variety of critical aspects in the cell environment during imaging, including the best choice of objective lenses, imaging system optimization, and data analysis.

McNally, J. G., Karpova, T., Cooper, J. and Conchello, J. A.

Three-dimensional imaging by deconvolution microscopy.  Methods 19: 373-385 (1999).  In this excellent review, the authors discuss the principles of deconvolution microscopy, describe different computational approaches for deconvolution, and provide an overview of the interpretation of deconvolved images with a particular emphasis on what artifacts may arise.

Agard, D. A.

Optical Sectioning Microscopy: Cellular architecture in three dimensions.  Annual Review of Biophysics and Bioengineering 13: 191-219 (1984).  A quarter-century after first being published, this review article remains an important source for understanding the fundamental concepts of gathering three-dimensional images in optical microscopy. The author discusses image formation, the point spread function, the contrast transfer function, and deconvolution methods to recover in-focus information.

Murray, J. M., Appleton, P. L., Swedlow, J. R. and Waters, J. C.

Evaluating performance in three-dimensional fluorescence microscopy.  Journal of Microscopy 228: 390-405 (2007).  A comprehensive analysis of optical sectioning performance that compares confocal, spinning disk, and deconvolution microscopy. The authors discuss a variety of critical parameters for each technique and conduct measurements with a carefully defined test specimen. This article should be consulted when attempting to determine the optimum imaging technique for thick specimens.

Sarder, P. and Nehorai, A.

Deconvolution methods for 3-D fluorescence microscopy images.  IEEE Signal Processing Magazine 23: Issue 3, 32-45 (2006).  This review is a great start for beginners in the field and presents an overview of various deconvolution techniques. Included is a brief schematic description of the microscope system and point spread function, performance measures, and a summary of the numerical results using simulated and real data.

Shaw, P.

Deconvolution in 3-D optical microscopy.  The Histochemical Journal 26: 687-694 (1994).  This review article attempts to explain image deconvolution, including those obtained from the confocal microscope, in a non-technical manner. In addition to discussions of the various key aspects of deconvolution, the author also provides examples of the application of image deconvolution to both conventional and confocal fluorescence images.

Swedlow, J. R., Hu, K., Andrews, P. D., Roos, D. S. and Murray, J. M.

Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy.  Proceedings of the National Academy of Sciences, USA 99: 2014-2019 (2002).  Examining a fluorescent protein marker (tubulin) in living cells, the investigators conclude that widefield fluorescence microscopy coupled to deconvolution has significant advantages over laser scanning confocal microscopy for quantitative studies in weakly fluorescent structures.

Rizzuto, R., Carrington, W. and Tuft, R. A.

Digital imaging microscopy in living cells.  Trends in Cell Biology 8: 288-292 (1998).  The authors present a nicely illustrated introduction to deconvolution microscopy with fluorescent proteins in living cells. Among the topics discussed are the microscope configuration, acquiring images, image restoration, example applications, and a comparison of deconvolution and confocal microscopy.

Ronneberger, O., Baddeley, D., Scheipl, F., Verveer, P. J., Burkhardt, H., Cremer, C., Fahrmeir, L., Cremer, T. and Joffe, B.

Spatial quantitative analysis of fluorescently labeled nuclear structures: Problems, methods, pitfalls.  Chromosome Research 16: 523-562 (2008).  A brilliant review addressing one of the most interesting areas in cell biology. The article was prepared by a team uniting expertise in biology, microscopy, image analysis, and statistics. It provides an overview of data acquisition options, preprocessing, image processing, and statistical analysis.