Sub-10 nm fluorescence imaging

Sauer, Markus; Helmerich, Dominic A.; Beliu, Gerti; Taban, Danush; Meub, Mara; Kuhlemann, Alexander; Doose, Soeren; Streit, Marcel

doi:10.1101/2022.02.08.479592

Cited by 2 publications

(4 citation statements)

References 60 publications

(105 reference statements)

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“…SMLM) are negatively affected by the water environment. In contrast, technologies relying on fluorophore fluctuations profit from the expansion, as the fluorophores are spatially separated and can fluctuate independently 13 . b , Repeated imaging is performed (up to 3000 images), in any desired imaging system (confocal, epifluorescence, etc.…”

Section: Extended Data Figure Legendsmentioning

confidence: 99%

“…First, the achievable structural resolution is determined by the labeling density, which is limited by the size of the fluorescent probes (typically 1 nanometer or larger) 12 . Second, fluorophores can interact via energy transfer at distances below 10 nm, which results in accelerated photoswitching (blinking) and photobleaching, and thus in substantially lower localization probabilities 13 .…”

Section: Introductionmentioning

confidence: 99%

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Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

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Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

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Section: Extended Data Figure Legendsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

Self Cite

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show abstract

“…Recent studies on fluorescently labeled DNA origami structures − have shown to what degree some identical fluorophores interact when separated by distances ranging from 1 to 10 nm, leading to unique antibunching characteristics, improved photostability, and increased photoblinking . The statistical signatures of these phenomena have very recently been proposed as a tool to determine molecular distances below 10 nm . Furthermore, such experiments provide building blocks that may help in constructing models of interacting fluorophores.…”

Section: Introductionmentioning

confidence: 99%

“…40 The statistical signatures of these phenomena have very recently been proposed as a tool to determine molecular distances below 10 nm. 42 Furthermore, such experiments provide building blocks that may help in constructing models of interacting fluorophores. While antibunching experiments analyze interphoton arrival times, in this paper, we specifically investigate the effects of interactions on excited state lifetimes and excitation probabilities.…”

Section: ■ Introductionmentioning

confidence: 99%

Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores

et al. 2022

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Quantitative fluorescence analysis is often used to derive chemical properties, including stoichiometries, of biomolecular complexes. One fundamental underlying assumption in the analysis of fluorescence datawhether it be the determination of protein complex stoichiometry by super-resolution, or step-counting by photobleaching, or the determination of RNA counts in diffraction-limited spots in RNA fluorescence in situ hybridization (RNA-FISH) experimentsis that fluorophores behave identically and do not interact. However, recent experiments on fluorophore-labeled DNA origami structures such as fluorocubes have shed light on the nature of the interactions between identical fluorophores as these are brought closer together, thereby raising questions on the validity of the modeling assumption that fluorophores do not interact. Here, we analyze photon arrival data under pulsed illumination from fluorocubes where distances between dyes range from 2 to 10 nm. We discuss the implications of non-additivity of brightness on quantitative fluorescence analysis.

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Sub-10 nm fluorescence imaging

Cited by 2 publications

References 60 publications

Visualizing proteins by expansion microscopy

Visualizing proteins by expansion microscopy

Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores

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