Assessment of 3D MINFLUX data for quantitative structural biology in cells

Prakash, Kirti; Curd, Alistair

doi:10.1101/2021.08.10.455294

Cited by 6 publications

(8 citation statements)

References 7 publications

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“…Or to be precise, near-field fluorophore interactions that decrease the localization probability of fluorophores separated by less than 10 nm have not been considered so far. The resulting lower image quality sparked a debate about the potential of new structured illumination SMLM methods (8)(9)(10)(11)(12) for molecular resolution imaging (13) but the contradiction between high localization precision and low localization probability remained enigmatic. Our findings are also important for quantitative SMLM approaches.…”

Section: Discussion and Outlookmentioning

confidence: 99%

“…For instance, the use of sequential structured illumination in combination with single-molecule detection as used in MINFLUX, SIMPLE and SIMFLUX allowed to improve the localization precision of direct stochastic optical reconstruction microscopy ( d STORM)( 6,7 ) using the red-absorbing cyanine dyes Alexa Fluor 647 (AF647) and Cy5 in photoswitching buffer to the 1-5 nm range( 8–12 ). Such high localization precisions permitted to resolve some fluorophores separated by only 6 nm on DNA origami and ~10 nm in nuclear pore complexes (NPCs), respectively ( 8,12 ).However, the results also sparked a debate about the spatial resolution claimed and the reliability of the method ( 13 ). In particular, the images revealed a low detection probability of fluorophores when separated by only a few nanometers evidenced by a high number of incomplete DNA origami and missing protein localizations in the biological samples.…”

Section: Main Textmentioning

confidence: 99%

“…Such high localization precisions permitted to resolve some fluorophores separated by only 6 nm on DNA origami and ~10 nm in nuclear pore complexes (NPCs), respectively (8,12). However, the results also sparked a debate about the spatial resolution claimed and the reliability of the method (13). In particular, the images revealed a low detection probability of fluorophores when separated by only a few nanometers evidenced by a high number of incomplete DNA origami and missing protein localizations in the biological samples.…”

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Sub-10 nm fluorescence imaging

Sauer

Helmerich

Beliu

et al. 2022

Preprint

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Advances in superresolution microscopy demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. Here, we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.

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Section: Discussion and Outlookmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

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confidence: 99%

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Sub-10 nm fluorescence imaging

Sauer

Helmerich

Beliu

et al. 2022

Preprint

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“…For a more in-depth explanation of their functioning, we would like to refer to recent reviews [48][49][50]. Readers are also pointed to the following for further reference on this topic [51][52][53][54][55].…”

Section: A Brief History Of Super-resolution Microscopymentioning

confidence: 99%

Super-resolution microscopy: a brief history and new avenues

Prakash

Diederich

Heintzmann

et al. 2022

Phil. Trans. R. Soc. A.

Self Cite

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Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below the classical diffraction limit of optical resolution. Acknowledged with the Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied in biomedical research, all with specific strengths and shortcomings. While some techniques are capable of nanometre-scale molecular resolution, others are geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on the latest trends in the field. We start with a brief historical overview of both conceptual and commercial developments. Next, we highlight important parameters for imaging successfully with a particular super-resolution modality. Finally, we discuss the importance of reproducibility and quality control and the significance of open-source tools in microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

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“…On theoretical grounds, this is to be expected because the donut illumination used in Minflux may also be regarded as some type of patterned illumination. However, as shown for Minflux [61], many additional problems have to be overcome until such a high localization precision can practically be converted into a similarly excellent structural resolution. Nonetheless, the improved localization precision obtainable by patterned illumination schemes may be highly useful and even necessary in case fluorescence photon yields are too low to allow the desired level of analysis.…”

Section: Position Measurement Using Smi Microscopymentioning

confidence: 99%

Spatially modulated illumination microscopy: application perspectives in nuclear nanostructure analysis

Cremer

Birk

2022

Phil. Trans. R. Soc. A.

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Thousands of genes and the complex biochemical networks for their transcription are packed in the micrometer sized cell nucleus. To control biochemical processes, spatial organization plays a key role. Hence the structure of the cell nucleus of higher organisms has emerged as a main topic of advanced light microscopy. So far, a variety of methods have been applied for this, including confocal laser scanning fluorescence microscopy, 4Pi-, STED- and localization microscopy approaches, as well as (laterally) structured illumination microscopy (SIM). Here, we summarize the state of the art and discuss application perspectives for nuclear nanostructure analysis of spatially modulated illumination (SMI). SMI is a widefield-based approach to using axially structured illumination patterns to determine the axial extension (size) of small, optically isolated fluorescent objects between less than or equal to 200 nm and greater than or equal to 40 nm diameter with a precision down to the few nm range; in addition, it allows the axial positioning of such structures down to the 1 nm scale. Combined with SIM, a three-dimensional localization precision of less than or equal to 1 nm is expected to become feasible using fluorescence yields typical for single molecule localization microscopy applications. Together with its nanosizing capability, this may eventually be used to analyse macromolecular complexes and other nanostructures with a topological resolution, further narrowing the gap to Cryoelectron microscopy. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

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Assessment of 3D MINFLUX data for quantitative structural biology in cells

Cited by 6 publications

References 7 publications

Sub-10 nm fluorescence imaging

Sub-10 nm fluorescence imaging

Super-resolution microscopy: a brief history and new avenues

Spatially modulated illumination microscopy: application perspectives in nuclear nanostructure analysis

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