Artifact-free high-density localization microscopy analysis

Marsh, Richard J.; Pfisterer, Karin; Bennett, Pauline M.; Hirvonen, L; Gautel, Mathias; Jones, Gareth E.; Cox, Susan

doi:10.1038/s41592-018-0072-5

Cited by 88 publications

(103 citation statements)

References 25 publications

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“…Certain chromatin structures could be observed for ~ 1 s, which underwent conformational changes during this period (Figure 1E). The spatial resolution at which structural elements can be observed (Materials and Methods) in time-resolved super-resolution data of chromatin was 63 ± 2 nm (Figure 1E), slightly more optimistic than the theoretical prediction (Figure 1B) ( 23 ). Thus, Deep-PALM identifies spatially heterogeneous coverage of chromatin as previously reported ( 10 , 13 , 14 , 16 , 22 ).…”

Section: Resultsmentioning

confidence: 74%

Coupling chromatin structure and dynamics by live super-resolution imaging

Barth

Bystricky

Shaban

2019

Preprint

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19Chromatin conformation regulates gene expression and thus constant remodeling of chromatin 20 structure is essential to guarantee proper cell function. To gain insight into the spatio-temporal 21 organization of the genome, we employ high-density photo-activated localization microscopy and 22 deep learning to obtain temporally resolved super-resolution images of chromatin in vivo. In 23 combination with high-resolution dense motion reconstruction, we confirm the existence of 24 elongated ~ 45 to 90 nm wide chromatin 'blobs', which appear to be dynamically associating 25 chromatin fragments in close physical and genomic proximity and adopt TAD-like interactions in 26 the time-average limit. We found the chromatin structure exhibits a spatio-temporal correlation 27 extending ~ 4 μm in space and tens of seconds in time, while chromatin dynamics are correlated 28 over ~ 6 μm and outlast 40 s. Notably, chromatin structure and dynamics are closely interrelated, 29 which may constitute a mechanism to grant access to regions with high local chromatin 30 concentration. 32 The three-dimensional organization of the eukaryotic genome plays a central role in gene regulation 33 (1-3). Its spatial organization has been prominently characterized by molecular and cellular 34 approaches including high-throughput chromosome conformation capture (Hi-C) (4) and 35 fluorescent in situ hybridization (FISH) (5). Topologically associated domains (TADs), genomic 36 regions that display a high degree of interaction, were revealed and found to be a key architectural 37 feature (6). Direct 3D localization microscopy of the chromatin fiber at the nanoscale (7) confirmed 38 the presence of TADs in single cells but also, among others, revealed great structural variation of 39 chromatin architecture (8, 9). To comprehensively resolve the spatial heterogeneity of chromatin, 40 super-resolution microscopy must be employed. Previous work showed that nucleosomes are 41 distributed as segregated, nanometer-sized accumulations throughout the nucleus (10-13) and that 42 the epigenetic state of a locus has a large impact on its folding (14, 15). However, to resolve the 43 fine structure of chromatin, high labeling densities, long acquisition times and, often, cell fixation 44 are required. This precludes capturing dynamic processes of chromatin in single live cells, yet 45 chromatin moves at different spatial and temporal scales. 46The first efforts to relate chromatin organization and its dynamics were made using a combination 47 of Photo-activated Localization Microscopy (PALM) and tracking of single nucleosomes (16). It 48 could be shown that nucleosomes mostly move coherently with their underlying domains, in 49 accordance with conventional microscopy data (17); however, a quantitative link between the 50 observed dynamics and the surrounding chromatin structure could not yet be established in real-51 time. Although it is becoming increasingly clear that chromatin motion and long-range interactions 52 are key to genome o...

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Section: Resultsmentioning

confidence: 74%

Coupling chromatin structure and dynamics by live super-resolution imaging

Barth

Bystricky

Shaban

2019

Preprint

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“…The 120 nm resolution of the RIM movie revealed actin filaments 214 linking two actin cores, in agreement with observations by electron microscopy ( Figure 3B). The robust 215 estimation of podosome surpassed that obtained with live-SIM (van den Dries et al, 2019;Meddens et al, 216 2016) or other computational methods (Marsh et al, 2018). Notably, the photobleaching and toxicity of RIM 217 was comparable to that of SIM ( Figure S4D) and podosome dynamics could be observed during 20 min 218 without detectable alteration of their reorganization.…”

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confidence: 89%

“…As seen in Figure 2B, RIM transverse resolution was much better 167 than that of confocal microscopy and similar to that of STED microscopy, about 120 nm (with fluorophores 168 emitting at 700 nm and a NA of 1.49). Interestingly, RIM reconstruction was free from common artefacts 169 such as the disappearance or merging of filaments (Marsh et al, 2018) and it provided the same image as 170 STED both in the dense and sparse regions of the sample. A total of 1 kW/cm², five times less than that 171 required for confocal microscopy, was delivered to the entire volume (30 µm x 30 µm field of view) in less 172 than 3 seconds.…”

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confidence: 99%

“…Importantly, the reconstruction scheme does not use any 381 regularisation except for the one needed for stabilizing the solution with respect to noise. As a result, 382 algoRIM is successful on both dense and sparse fluorescent samples and avoids the common artefacts 383 encountered in fluctuation or sparsity-based microscopy such as the over or underestimation of strongly or 384 weakly labelled features (Marsh et al, 2018). Hence, as compared with the SEM image, RIM showed more 385 details than dSTORM of the densely fluorescent podosome nodes ( Figure 1C), and when the same vimentin 386 filaments were observed by RIM or by STED microscopy, the same image was obtained at the same 387 resolution ( Figure 2B), which underscored the fidelity of RIM image reconstruction.…”

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confidence: 99%

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Super-resolved live-cell imaging using Random Illumination Microscopy

Mangeat

Labouesse

Allain

et al. 2020

Preprint

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25Super-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-26 induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups 27 curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging 28 reaching a 114nm/8Hz resolution at 30 µm depth. Random Illumination Microscopy (RIM) consists in 29 shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the 30 variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. 31 Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and 32 ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image 33 molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range 34 of biological situations such as the motion of Myosin II minifilaments in Drosophila. 35 36 37 38

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“…This demonstrates that PA-SiR-Halo enables live-cell SMLM of intracellular targets. 38 was used to achieve imaging at high emitter densities to capture fast structural changes. b, Sum projection over the first 10 s mimicking the diffraction limited image.…”

Section: Live-cell Single-particle Tracking and Smlmmentioning

confidence: 99%

Photoactivation of silicon rhodamines via a light-induced protonation

Frei

Hoess

Lampe

et al. 2019

Preprint

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We present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired sideproducts released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

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Artifact-free high-density localization microscopy analysis

Cited by 88 publications

References 25 publications

Coupling chromatin structure and dynamics by live super-resolution imaging

Coupling chromatin structure and dynamics by live super-resolution imaging

Super-resolved live-cell imaging using Random Illumination Microscopy

Photoactivation of silicon rhodamines via a light-induced protonation

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