4Pi-Confocal Microscopy Provides Three-Dimensional Images of the Microtubule Network with 100- to 150-nm Resolution

Nagorni, Matthias; Hell, Stefan W.

doi:10.1006/jsbi.1998.4037

Cited by 70 publications

(41 citation statements)

References 24 publications

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“…For example, 4Pi-microscopy and STED-microscopy have been used to study nanostructures on the cell membrane [Bahlmann et al 2001;Glaschick et al 2007;Perinetti et al 2009], of the cytoplasm [Egner et al 1998;Hell and Nagorni 1998;Nagorni and Hell 1998;Schrader et al 1998;Gugel et al 2004;Egner et al 2005] as well of the nucleus [Bewersdorf et al 2006;Lang et al 2010] and of the nuclear envelope [Hüve et al 2008;Baddeley et al 2006]. In addition, STED microscopy has been shown to be useful for neurobiology in the study of synaptic connections between nerve cells [Willig et al 2006;Nagerl et al 2008].…”

Section: Impact Of Enhanced Resolution Light Microscopy In the Bioscimentioning

confidence: 99%

Resolution enhancement techniques in microscopy

Cremer

Masters

2013

EPJ H

146

133

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Abstract. We survey the history of resolution enhancement techniques in microscopy and their impact on current research in biomedicine. Often these techniques are labeled superresolution, or enhanced resolution microscopy, or light-optical nanoscopy. First, we introduce the development of diffraction theory in its relation to enhanced resolution; then we explore the foundations of resolution as expounded by the astronomers and the physicists and describe the conditions for which they apply. Then we elucidate Ernst Abbe's theory of optical formation in the microscope, and its experimental verification and dissemination to the world wide microscope communities. Second, we describe and compare the early techniques that can enhance the resolution of the microscope. Third, we present the historical development of various techniques that substantially enhance the optical resolution of the light microscope. These enhanced resolution techniques in their modern form constitute an active area of research with seminal applications in biology and medicine. Our historical survey of the field of resolution enhancement uncovers many examples of reinvention, rediscovery, and independent invention and development of similar proposals, concepts, techniques, and instruments. Attribution of credit is therefore confounded by the fact that for understandable reasons authors stress the achievements from their own research groups and sometimes obfuscate their contributions and the prior art of others. In some cases, attribution of credit is also made more complex by the fact that long term developments are difficult to allocate to a specific individual because of the many mutual connections often existing between sometimes fiercely competing, sometimes strongly collaborating groups. Since applications in biology and medicine have been a major driving force in the development of resolution enhancing approaches, we focus on the contribution of enhanced resolution to these fields. a

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Section: Impact Of Enhanced Resolution Light Microscopy In the Bioscimentioning

confidence: 99%

Resolution enhancement techniques in microscopy

Cremer

Masters

2013

EPJ H

146

133

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“…In addition to its obvious advantages to confocal fluorescence microscopy, the glycerol objective lens is of particular relevance to 4 Pi-confocal microscopy of fixed specimens (Hell et al, 1997;Nagorni & Hell, 1998) and to related concepts using two opposing lenses, such as the I 5 M (Gustafsson et al, 1999;Nagorni & Hell, 2001). In the case of the 4 Pi-confocal microscope, the spherical aberration induced by the RI mismatch between the oil/glass system and the glycerol mountant limits the object thickness to 10 -20 µm.…”

Section: Discussionmentioning

confidence: 99%

A new high‐aperture glycerol immersion objective lens and its application to 3D‐fluorescence microscopy

Martini

Bewersdorf

Hell

2002

Journal of Microscopy

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SummaryHigh-resolution light microscopy of glycerol-mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of ~1.51 is close to that of ~1.45 of the glycerol mountant, so that refractive index mismatchinduced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass-corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi-aperture angle of 68.5°, which is slightly larger than that of the diffraction-limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 µm thickness and for a 80% glycerol-water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch-induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed.

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“…e 4Pi image after deconvolution design (Gustafsson 1999). One of the major applications of 4Pi and I5M microscopy has been the study of subcellular organelles in 3D (Nagorni and Hell 1998;Gustafsson 1999;Egner et al 2004;Medda et al 2006;Ivanchenko et al 2007). These two concepts have subsequently been combined with techniques that improve the lateral resolution so as to improve the resolution in all three dimensions, 4Pi-STED (Dyba et al 2003) and I5S ), as will be discussed below.…”

Section: Microscopy Designs and Confocal Techniquesmentioning

confidence: 99%