Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

doi:10.1364/boe.7.002163

Cited by 26 publications

(17 citation statements)

References 19 publications

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“…(112) A drawback of multifocal methods that span a large axial range is inefficient use of signal, especially at the extremes of the focal stack, where much of the signal is split into planes that are too out of focus to provide useful information. To improve the 3D precision attainable from each plane, multifocal imaging has been combined with astigmatism (see section 3.2),(114, 115) and we anticipate the possibility of further interesting modifications to multifocus techniques.…”

Section: Methods For 3d Localizationmentioning

confidence: 99%

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

2017

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Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.

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Section: Methods For 3d Localizationmentioning

confidence: 99%

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

2017

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“…TADs (Boettiger et al 2016;Szabo et al 2018 (Boettiger et al 2016;Szabo et al 2018;Oudjedi et al 2016) . However, direct, single-cell, super-resolved visualization of putative phase-separated factors would be needed to directly establish whether these proteins can form spherical objects.…”

Section: Simultaneousmentioning

confidence: 99%

TADs or no TADS: Lessons From Single-cell Imaging of Chromosome Architecture

Gizzi

Cattoni

Nöllmann

2020

Journal of Molecular Biology

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Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulate their function. Genome-wide studies revealed a new level of organization at the kilobase-to-Megabase scale termed "topological associating domains" (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one "functional unit". Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments and propose an interpretation reconciling population-based findings with direct single cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.

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“…43 A key benefit of MUM is that it can also be used to image extended objects, 41,44 whereas the emphasis for PSF-engineering has been on localization of PSFs that are the images of pointlike objects. 45,46 There is also the opportunity to use MUM/MFM in combination with engineered PSFs for even better localization precision, as has been done using an astigmatic PSF [47][48][49] and an Airy-beam-based PSF. 50,51…”

Section: A Multifocal-plane Microscopy (Mum)mentioning

confidence: 99%

Advances in 3D single particle localization microscopy

et al. 2019

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The spatial resolution of conventional optical microscopy is limited by diffraction to transverse and axial resolutions of about 250 nm, but localization of point sources, such as single molecules or fluorescent beads, can be achieved with a precision of 10 nm or better in each direction. Traditional approaches to localization microscopy in two dimensions enable high precision only for a thin in-focus layer that is typically much less than the depth of a cell. This precludes, for example, super-resolution microscopy of extended three-dimensional biological structures or mapping of blood velocity throughout a useful depth of vasculature. Several techniques have been reported recently for localization microscopy in three dimensions over an extended depth range. We describe the principles of operation and typical applications of the most promising 3D localization microscopy techniques and provide a comparison of the attainable precision for each technique in terms of the Cramér-Rao lower bound for high-resolution imaging.

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Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

Cited by 26 publications

References 19 publications

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

TADs or no TADS: Lessons From Single-cell Imaging of Chromosome Architecture

Advances in 3D single particle localization microscopy

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