Superresolution microscopy with transient binding

Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt‐Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

doi:10.1016/j.copbio.2015.12.009

Cited by 21 publications

(18 citation statements)

References 53 publications

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“…Furthermore, photobleaching of tagged fluorescent molecules limits measurement time and prevents long‐term imaging of targets. Recently, following the development of PAINT, binding‐activated or transiently binding probes have expanded the scope of SR imaging to functional studies . When in the immediate vicinity of their target, these probes either become fluorescent, temporarily bind to the target, or both, thereby creating a “flash” of fluorescence that is used to locate the target of interest.…”

Section: Figurementioning

confidence: 99%

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

et al. 2018

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Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-β aggregation, as well as the structural remodeling of amyloid-β fibrils by the compound epi-gallocatechin gallate.

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Section: Figurementioning

confidence: 99%

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

et al. 2018

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“…3 shows that the effective free imager concentration can be reduced both by adding the high-affinity P1+ quencher strand www.theNanoResearch.com|www.Springer.com/journal/12274 | Nano Research Nano Res. list includes the quencher strand designs shown in this manuscript and extends to the potential use of molecular beacon imagers [21]. Nevertheless, the use of quenchers shown here is a practical alternative to actually removing imagers from the solution as we further demonstrate below in experiments with biological samples.…”

Section: Tuning Of the Binding-event Rate And Background Fluorescencementioning

confidence: 99%

Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT

et al. 2018

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The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ~10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA "imager" strands bind transiently and with high specificity to complementary target "docking" strands anchored to the structure of interest. The localization of single binding events enables the assembly of a superresolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce QuencherExchange-PAINT-a new approach to Exchange-PAINT in regular open-top imaging chambers-which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses "quencher" strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized QuencherExchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps.

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“…Multi-color STORM is not as straightforward, as it is a challenge to find two distinct fluorophores that have good blinking characteristics in the same environment, and inducing photoswitching of fluorophores outside of the far-red channel usually require high laser power illumination [21,22]. In DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) [23,24], blinking is generated by the transient hybridization of short DNA single-strand coupled to a fluorophore (imager strand) with its complementary strand (docking strand) attached to an antibody targeting the structure of interest [25,26]. DNA-PAINT does not require a specific buffer or very high laser power, as the blinking does not depend on the photophysics of the fluorophore.…”

Section: Introductionmentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2020

Methods

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Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a prerequisite for meaningful biological discovery.

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Superresolution microscopy with transient binding

Cited by 21 publications

References 53 publications

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT

About samples, giving examples: Optimized Single Molecule Localization Microscopy

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