Whole-Cell, 3D, and Multicolor STED Imaging with Exchangeable Fluorophores

Spahn, Christoph; Grimm, Jonathan B.; Lavis, Luke D.; Lampe, Marko; Heilemann, Mike

doi:10.1021/acs.nanolett.8b04385

Cited by 116 publications

(141 citation statements)

References 25 publications

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“…Consequently, we were able to image the same structures across multiple frames without noticeable reduction in intensity as well as cell viability. In this context, the use of diffusing fluorescent lipid analogs offers a robust alternative to other labelling strategies using exchangeable fluorophores 33 .…”

Section: Resultsmentioning

confidence: 99%

“…A different depletion scheme can be used to increase mostly the axial resolution, relying on a "bottle-shaped" beam for depletion (we will call it z-STED hereafter) 28 . The z-STED depletion pattern has been used both alone and together with 2D STED (3D STED) for imaging [29][30][31][32][33][34][35] and STED-FCS [36][37][38] in solution or cytoplasm. However, the exacerbated sensitivity to aberrations 39,40 and difficulty of operation of the z-STED depletion pattern have prevented its widespread use.…”

Section: Introductionmentioning

confidence: 99%

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z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

Barbotin

Urbančič

Galiani

et al. 2019

Preprint

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AbstractSuper-resolution STED microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy/2D) or axially (z/3D). 2D STED has been extensively used to elucidate the nanoscale membrane structure and dynamics, via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of approximately 100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

Barbotin

Urbančič

Galiani

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…In order to be suitable for STED microscopy, a high labeling density is required to saturate all target binding sites . Higher concentrations of exchangeable fluorophore labels can achieve such a pseudo‐permanent labeling and enable STED imaging with minimized photobleaching . DNA‐PAINT labels have been previously combined with STED microscopy using longer oligonucleotides for stable hybridization and denaturing washing buffers to exchange the labels between imaging rounds …”

Section: Figurementioning

confidence: 99%

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Spahn

Hurter²,

Glaesmann

et al. 2019

Angew Chem Int Ed

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Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.

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“…Various solutions to minimize photobleaching in STED microscopy have been introduced, including dynamic tuning of the excitation light during image acquisition, the development of photostable fluorophores, or the use of fluorophores with multiple off‐states . An alternative route is using fluorophore labels that reversibly bind to a target structure and exchange with a reservoir, making STED microscopy insensitive to photobleaching and enabling multicolor and 3D imaging of whole cells . This is achieved by a permanent exchange of labels, which removes photobleached fluorophores and replenishes them with intact ones that are present in the imaging buffer.…”

Section: Figurementioning

confidence: 99%

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Spahn

Hurter²,

Glaesmann

et al. 2019

Angewandte Chemie

Self Cite

View full text Add to dashboard Cite

Photobleaching is amajor challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image.P hotobleaching can be bypassed by using exchangeable labels,w hich transiently bind to and dissociate from at arget, thereby replenishing the destroyed labels with intact ones from areservoir.Here,wedemonstrate confocal and STED microscopyw ith short, fluorophorelabeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels.W es how that this concept is suitable for targeted, two-color STED imaging of whole cells.

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Whole-Cell, 3D, and Multicolor STED Imaging with Exchangeable Fluorophores

Cited by 116 publications

References 25 publications

z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

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