Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Biophysical Journal
Hilmar GugelStefan W Hell

Abstract

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and li...Continue Reading

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Citations

May 8, 2008·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Peter M Carlton
Dec 1, 2007·Journal of Biological Physics·Sylvia GlaschickG Ulrich Nienhaus
Apr 9, 2005·Trends in Cell Biology·Alexander Egner, Stefan W Hell
May 20, 2008·Nature Methods·Roman SchmidtStefan W Hell
Nov 18, 2006·Proceedings of the National Academy of Sciences of the United States of America·Jörg BewersdorfKendall L Knight
Jun 27, 2009·Annual Review of Neuroscience·Brian A WiltMark J Schnitzer
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Jun 16, 2006·Journal of Microscopy·J BewersdorfS W Hell
Oct 7, 2004·Current Opinion in Neurobiology·Stefan W HellStefan Jakobs
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Feb 5, 2010·Journal of Cell Science·Marion LangKarsten Rippe

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