Abstract
Conventional microscopy techniques, namely, the confocal microscope or deconvolution processes, are resolution limited to approximately 200–250 nm by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20–50 nm in diameter. In the mid-2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular co-localization questions. This “breaking” of the diffraction barrier, affording resolution beyond 200 nm, is termed super-resolution microscopy. More recent approaches include single-molecule localization (such as photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) and point spread function engineering (such as stimulated emission depletion (STED) microscopy). In this review, we explain basic principles behind currently commercialized super-resolution setups and address advantages and considerations in applying these techniques to protein co-localization in biological systems.
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Acknowledgments
We greatly appreciate the willingness of Applied Precision, Inc., a division of GE, Vutara, and Carl Zeiss to test samples on their instruments and discuss the implementation of technique. Work in the Storrie laboratory was supported by NIH grants, R01GM092960 and R01HL119393. Work in the Baldini laboratory is supported by NIH grant, R01DK080424.
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MacDonald, L., Baldini, G., Storrie, B. (2015). Does Super-Resolution Fluorescence Microscopy Obsolete Previous Microscopic Approaches to Protein Co-localization?. In: Tang, B. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 1270. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2309-0_19
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DOI: https://doi.org/10.1007/978-1-4939-2309-0_19
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