Estimating the localization spread function of static single-molecule localization microscopy images

Fazekas, F.; Kim, Sumin; Flanagan-Natoli, Jennifer C; Sumrall, Emily R; Veatch, Sarah L.

doi:10.1016/j.bpj.2022.06.036

Cited by 5 publications

(6 citation statements)

References 38 publications

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“…Neurons were washed three times in PBS and stained with goat-anti-rabbit Alexa Fluor 647 Fab Fragment (1:800; Jackson ImmunoResearch 111-607-003) for an hour, washed three times with PBS, and imaged. Imaging and processing to determine localization and correct drift were conducted as previously described 99 . Samples were imaged in a buffer containing 100mM Tris, 25mM NaCl, 10% glucose, 1% bmercaptoethanol, 500 μg/ml glucose oxidase (Sigma) and 80 μg/ml catalase (Sigma).…”

Section: Dstorm Sample Preparation and Imagingmentioning

confidence: 99%

“…Individual NPCs were segmented through a multi-step and fully automated process designed to identify objects of a particular size and aspect ratio while not biasing towards particles with distinct pores. First, an initial segmentation was conducted using a home-built implementation of DBSCAN 99,104 , with ε=40 nm and minPts = 25. The size of the major and minor axes of segmented objects were then determined by tabulating the eigenvalues of the covariance of x,y points with each segment.…”

Section: Npc Segmentation From Dstormmentioning

confidence: 99%

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TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis

Kim

Phan

Ellisman

et al. 2023

Preprint

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Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss of function causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs.

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Section: Dstorm Sample Preparation and Imagingmentioning

confidence: 99%

Section: Npc Segmentation From Dstormmentioning

confidence: 99%

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis

Kim

Phan

Ellisman

et al. 2023

Preprint

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“…This can be accomplished using the MATLAB function spacetime_xcor() posted in the SMLM-analysis distribution. This base function includes an efficient code to tabulate the pairwise distances between localizations over time, adapted from [42] and described previously [41], as well as boundary conditions for arbitrary spatial ROIs and temporal windows, as described [34]. In some cases, long-range gradients in images can obscure results when an analysis of short-range correlations are desired.…”

Section: Methodsmentioning

confidence: 99%

“…Autocorrelation functions provide information on how labeled proteins move and how they selfassociate, while cross-correlation functions probe the colocalization of proteins, as well as the dynamics and effective energy of these associations [15,[28][29][30][31][32][33]. Pair-correlation methods can also be used as part of a general image processing pipeline to enhance or characterize SMLM datasets [34,35].…”

Section: Introductionmentioning

confidence: 99%

Measuring the co-localization and dynamics of mobile proteins in live cells undergoing signaling responses

Shelby

Veatch

2022

Preprint

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Single molecule imaging in live cells enables the study of protein interactions and dynamics as they participate in signaling processes. When combined with fluorophores that stochastically transition between fluorescent and reversible dark states, as in super-resolution localization imaging, labeled molecules can be visualized in single cells over time. This improvement in sampling enables the study of extended cellular responses at the resolution of single molecule localization. This chapter provides optimized experimental and analytical methods used to quantify protein interactions and dynamics within the membranes of adhered live cells. Importantly, the use of pair-correlation functions resolved in both space and time allows researchers to probe interactions between proteins on biologically relevant distance and time-scales, even though fluorescence localization methods typically require long times to assemble well-sampled reconstructed images. We describe an application of this approach to measure protein interactions in B cell receptor signaling and include sample analysis code for post-processing of imaging data. These methods are quantitative, sensitive, and broadly applicable to a range of signaling systems.

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“…Sauer and colleagues recently summarized the three key factors determining image resolution in SMLM: i) localization precision (statistical scattering of the measured position coordinates), ii) localization accuracy (systematic deviation between the measured and true position) and iii) the labeling density ( Helmerich et al, 2022 ). The first of these factors has been the main focus of efforts for improving image resolution, via hardware, e.g., by combining improved structured illumination conditions with single-molecule detection ( Kalisvaart et al, 2022 ; Zhan et al, 2022 ), DNA-PAINT with STORM ( Cnossen et al, 2020 ), using cryo-SMLM and ad hoc software corrections ( Schneider et al, 2020 ; Hinterer et al, 2022 ), or via computational methods that enhance localization precision through engineering of the PSF or other approaches as applied to a single ( Shaw et al, 2022 ) ( Li M. et al, 2022 ) or multiple fluorophores (colocalization precision) ( Koyama-Honda et al, 2005 ; Donnert et al, 2007 ; Lemmer et al, 2009 ; Curthoys et al, 2013 ; Weisenburger et al, 2014 ; Georgieva et al, 2016 ; Willems and MacGillavry, 2022 ).…”

Section: Future Prospectsmentioning

confidence: 99%

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

Barrantes

2022

Front. Mol. Biosci.

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Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm–10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

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Estimating the localization spread function of static single-molecule localization microscopy images

Cited by 5 publications

References 38 publications

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis

TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis

Measuring the co-localization and dynamics of mobile proteins in live cells undergoing signaling responses

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

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