4Pi-microscopy of the Golgi apparatus in live mammalian cells

Egner, Alexander; Verrier, Sophie; Goroshkov, Alexander V.; Soling, H.; Hell, Stefan W.

doi:10.1016/j.jsb.2003.10.006

Cited by 69 publications

(49 citation statements)

References 19 publications

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“…5). Imaging nuclear structures with interference microscopy techniques such as 4Pi microscopy is potentially difficult: Wavefront distortions in the vicinity of cell nuclei that degrade the 4Pi PSF have been reported (15). However, by matching the cytosolic and nuclear refractive indices by glycerol-embedding the sample and using 1.35 NA glycerol immersion lenses (30) with the embedding medium for immersion, we found that refractive indices could be matched for HeLa cells.…”

Section: Methodsmentioning

confidence: 89%

“…HeLa cells were fixed and stained for ␥-H2AX and H2AX at 15, 45, 90, 180, 360, and 720 min after exposure to 3 Gy ionizing radiation (IR). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, it has been impossible to distinguish f luorescent signals in a threedimensional (3D) environment that are closer together than 500-800 nm in distance given the depth resolution limits of current light microscopes. With Ϸ100-nm resolution along the optic axis (z axis), 4Pi microscopy (13,14) provides a significant increase in resolution and has allowed more defined images of cellular structures such as microtubules, mitochondria, or the Golgi apparatus (15). However, until now, imaging of endogenous nuclear proteins had not been achieved (a comparison of 4Pi vs. confocal is described in Materials and Methods).…”

mentioning

confidence: 99%

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H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

Bewersdorf

Bennett

Knight

2006

Proc. Natl. Acad. Sci. U.S.A.

115

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DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form ␥-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that ␥-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of ␥-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of ␥-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.␥-H2AX ͉ H2AX chromatin clusters ͉ super-resolution 4Pi microscopy ͉ chromatin response to DNA damage ͉ 3D quantification of chromatin structures M aintenance of genome integrity is critical for organism development and survival, and higher organisms have evolved sophisticated mechanisms for detection and repair of chromosome breaks. DNA damage results in the rapid and coordinated action of various pathways, including activation of cell cycle checkpoints (1, 2), histone modification near the site of the break (3, 4), and recruitment of chromatin remodeling enzymes (4-6), cohesins (7-9), and DNA repair proteins (1, 2, 10). The significance of these molecular processes is highlighted by the fact that defects in many are associated with an increased risk of cancer and developmental and immunologic abnormalities (1). Important insights into the positioning of nuclear signaling and repair proteins and their response to various types and levels of genomic insults have been achieved by using immunofluorescence methods (10-12). However, it has been impossible to distinguish f luorescent signals in a threedimensional (3D) environment that are closer together than 500-800 nm in distance given the depth resolution limits of current light microscopes. With Ϸ100-nm resolution along the optic axis (z axis), 4Pi microscopy (13, 14) provides a significant increase in resolution and has allowed more defined images of cellular structures such as microtubules, mitochondria, or the Golgi apparatus (15). However, until now, imaging of endogenous nuclear proteins had not been achieved (a comparison of 4Pi vs. confocal is described in Materials and Methods). In this study, we describe the successful use of 4Pi microscopy to visualize endogenous nuclear proteins. By applying previo...

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Section: Methodsmentioning

confidence: 89%

“…HeLa cells were fixed and stained for ␥-H2AX and H2AX at 15, 45, 90, 180, 360, and 720 min after exposure to 3 Gy ionizing radiation (IR). Fig.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

Bewersdorf

Bennett

Knight

2006

Proc. Natl. Acad. Sci. U.S.A.

115

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“…A notable exception, though, was (diffraction-limited) 4Pi microscopy yielding superresolution of dynamic living samples along the optic axis [15][16][17][18]. While the possibility of overcoming the diffraction limit in living cells was demonstrated a decade ago [19], it was not before 2007 that dynamic imaging with nanoscale resolution in the focal plane was demonstrated at 80 frames per second [20] and in 2008 that video-rate imaging was achieved in living cells [21].…”

Section: Biophotonicsmentioning

confidence: 99%

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

Lauterbach

Keller

Schönle

et al. 2010

Journal of Biophotonics

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We compare the performance of video‐rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4‐fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video‐rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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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%