Template-free 2D particle fusion in localization microscopy

Heydarian, Hamidreza; Schueder, Florian; Strauss, Maximilian T.; Werkhoven, Ben van; Fazel, Mohamadreza; Lidke, Keith A.; Jungmann, Ralf; Stallinga, Sjoerd; Rieger, Bernd

doi:10.1038/s41592-018-0136-6

Cited by 70 publications

(117 citation statements)

References 22 publications

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…This is similar to the error range of PAINT data at 30% DOL. Consistent with our previous work therefore, 12 we observe that STORM imaging requires a higher DOL than PAINT to achieve a similar performance. The simulations also indicate that a highquality reconstruction (error <10 nm) requires at least 50-100 particles (Figure 1d) for PAINT data with 50% DOL.…”

supporting

confidence: 92%

“…In our earlier work 12 , we kept all initially picked particles for the final super-particle. We only removed many of the bad registrations from the all-to-all matrix as long as the graph stays connected.…”

Section: Outlier Particle Removalmentioning

confidence: 99%

“…Our ground-truth model consists of 2x16 copies of NUP107 arranged in 8 pairs on the cytoplasmic and nuclear ring of the NPC with ~13 degree of azimuthal shift (Figure 1b). PAINT and STORM switching kinetics were simulated as earlier described 12 . For each parameter setting, we generated 15 datasets containing 100 particles each.…”

Section: Outlier Particle Removalmentioning

confidence: 99%

“…The processing pipeline is built upon our previous 2D method 12 with modifications to each step to handle 3D localizations (Figure 1a). Briefly, we first register all N segmented particles in pairs, which provides ( − 1)/2 relative registration parameters )* (3D rotation and translation from particle i to j).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

Heydarian

Przybylski²,

Schueder

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons. Main textSingle molecule localization microscopy (SMLM) is capable of resolving biological structure at the nanometer scale. However, SMLM image resolution is ultimately limited by the density of the fluorescent labels on the structure of interest and the finite precision of each localization 1, 2 . Recently, methods for obtaining higher precision localizations have been reported, which work by either increasing the number of collected photons per molecule via e.g. cryogenic imaging 3, 4 , or by introducing patterned illumination 5, 6 . The first limitation remains, however, and one approach to boosting the apparent degree of labeling (DOL) and filling in missing labels can be applied when the sample consists of many identical copies of the structure of interest (e.g. a protein complex). In this case, by combining many structures into a single "super-particle", the effective labelling density is increased, and the resulting super-particle has a high number of localizations leading to a significantly improved signal-to-noise ratio and resolution.Previous approaches to this problem can be classified as either template-based or adaptations of existing single particle analysis (SPA) algorithms originally developed for cryo-electron microscopy (EM). Template-based methods 7, 8 are computationally efficient, however, they are susceptible to template bias artefacts. Methods derived from SPA for cryo-EM have previously been adapted 9, 10 and employed to generate 3D volumes from 2D projection data. These approaches are, however, intrinsically 2D to 3D, as they assume that the raw data are projections. Recently, Shi et. al 11 also described a structure-specific method for 3D fusion, although they implicitly assume cylindrical particles and projected the volume onto top views only.

show abstract

supporting

confidence: 92%

Section: Outlier Particle Removalmentioning

confidence: 99%

Section: Outlier Particle Removalmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

Heydarian

Przybylski²,

Schueder

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…At this scale, colocalization does not exist and is replaced by the measurement of distances between the localizations of different species (Georgieva et al, 2016;Lagache et al, 2018;Pageon et al, 2016). A recent development in SMLM analysis is the use of single particle averaging to reach molecular details beyond image resolution by averaging multiple similar objects (Broeken et al, 2015;Heydarian et al, 2018;Laine et al, 2015;Salas et al, 2017;Sieben et al, 2018a). A detailed review of the available algorithms and software is beyond the scope of the present article, but we encourage the interested reader to explore this very dynamic area that constitutes the next logical step toward leveraging SMLM possibilities.…”

Section: Analysis Of Smlm Datamentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2019

Preprint

View full text Add to dashboard Cite

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 pre-requisite for meaningful biological discovery.

show abstract

Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy

Aktalay

Khan²,

Bossi

et al. 2023

Angewandte Chemie

View full text Add to dashboard Cite

New photoactivatable fluorescent dyes (rhodamine, carbo‐ and silicon‐rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2‐nitrobenzyloxycarbonyl unit with an alpha‐carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live‐cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live‐cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single‐digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

show abstract

Template-free 2D particle fusion in localization microscopy

Cited by 70 publications

References 22 publications

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy

Contact Info

Product

Resources

About