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Microscopy Reference Library

Optical Sectioning Microscopy

The ability to image thin sections without having to mechanically slice a thick specimen is afforded by optical sectioning microscopy. Sections are achieved by eliminating the excitation and detection of fluorescence that originates in regions removed from the focal plane. The reviews listed in this section should be available to students and investigators who have access to subscriptions through their host institutions.

Conchello, J. A. and Lichtman, J. W.

Optical sectioning microscopy.  Nature Methods 2: 920-931 (2005).  An excellent review that covers confocal principles, contrast, resolution, scanning implementations, spinning disk microscopy, deconvolution, potential artifacts, and alternative approaches to achieving and analyzing crisp optical sections.

Agard, D. A.

Optical sectioning microscopy: Cellular architecture in three dimensions.  Annual Review of Biophysics and Bioengineering 13: 191-219 (1984).  One of the original and most authoritative review articles describing optical sectioning techniques in the biological sciences. Among the topics covered are image formation theory, the contrast transfer function, point spread functions, Fourier transformation, and deconvolution.

Feng, D., Marshburn, D., Jen, D., Weinberg, R. J., Taylor, R. M. and Burette, A.

Stepping into the third dimension.  Journal of Neuroscience 27: 12757-12760 (2007).  The authors discuss recent advances in optical section microscopy along with novel fluorescent proteins and probes in applications that enable imaging of subcellular structures in space and time. Software useful for optical sectioning microscopy is also discussed.

Ferko, M. C., Patterson, B. W. and Butler, P. J.

High-resolution solid modeling of biological samples imaged with 3D fluorescence microscopy.  Microscopy Research and Technique 69: 648-655 (2006).  An extensive review of three-dimensional volume rendering using solid modeling algorithms for extended computational analysis. The authors discuss techniques to overcome axial distortions produced by refractive index mismatches.

McNally, J. G., Preza, C., Conchello, J. A. and Thomas, L. J.

Artifacts in computational optical-sectioning microscopy.  Journal of the Optical Society of America A11: 1056-1067 (1994).  Analysis of optical sectioning models to examine point spread function shift variance, nonlinear algorithmic approaches, and best-possible linear reconstruction of the specimen. Although an early paper in this field, the authors conducted an excellent study of problems associated with optical sectioning.

Paddock S.

Optical sectioning: Slices of life.  Science 295: 1319-1321 (2002).  Written for the layperson, this review contains excellent references and examples of optical sectioning using laser scanning confocal microscopy. The author discusses instrumentation, pitfalls, and emerging technologies that may prove beneficial in the future.

Wright, S. J. and Schatten G.

Confocal fluorescence microscopy and three-dimensional reconstruction.  Journal of Electron Microscopy Technique 18: 2-10 (1990).  Although dated, this review article discusses laser scanning and spinning disk optical sectioning techniques and volume rendering of the resulting image stacks. It is also an excellent starting point for novices in the field.

Verveer, P. J., Gemkow, M. J. and Jovin, T. M.

A comparison of image restoration approaches applied to three-dimensional confocal and widefield microscopy.  Journal of Microscopy 193: 50-61 (1999).  Exhaustive analysis of algorithms used to analyze (by deconvolution) optical sections obtained with laser scanning and deconvolution microscopy. The authors utilized simulations and real data to examine noise models to compare the methods.

Centonze, V. E. and White, J. G.

Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.  Biophysical Journal 75: 2015-2024 (1998).  One of the first comprehensive reviews of optical sectioning using multiphoton microscopy. The authors conduct side-by-side comparisons with laser scanning confocal instruments to demonstrate a significant advantage for multiphoton with regards to generating optical sections.

Egner A. and Hell, S. W.

Fluorescence microscopy with super-resolved optical sections.  Trends in Cell Biology 15: 207-215 (2005).  Optical sectioning with superresolution microscopy is becoming a topic of interest to a number of investigators. The authors describe a number of superresolution techniques and explore differences in the point spread function and capabilities of these instruments to generate ultrathin sections for three-dimensional reconstruction at much higher resolution than afforded by traditional microscopy techniques.