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Optical sectioning microscopy

Nature Methods volume 2, pages 920–931 (2005)Cite this article

Abstract

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.

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Figure 1: The confocal principle.
Figure 2: The PSF.
Figure 3: PSF, SNR and background rejection.
Figure 4: Linear unmixing.
Figure 5: Deconvolution results.

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Authors and Affiliations

  1. Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, 73104, Oklahoma, USA

    José-Angel Conchello

  2. Program in Biomedical Engineering, University of Oklahoma, Norman, 73019, Oklahoma, USA

    José-Angel Conchello

  3. Molecular and Cell Biology Department, Harvard University, Cambridge, 02138, Massachusetts, USA

    Jeff W Lichtman

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Correspondence to José-Angel Conchello.

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Conchello, JA., Lichtman, J. Optical sectioning microscopy. Nat Methods 2, 920–931 (2005). https://doi.org/10.1038/nmeth815

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