Accelerating iterative deconvolution and multiview fusion by orders of magnitude

Guo, Min; Y, Li; Y, Su; Lambert, Talley J.; Dd, Nogare; Mw, Moyle; Duncan, Leighton H.; Ikegami, Richard; Santella, Anthony; Rey-Suarez, Ivan; Green, D; Chen, J; Vishwasrao, Harshad D.; Ganesan, Sundar; Jc, Waters; Cm, Annunziata; Hafner, Markus; Mohler, WA; Ab, Chitnis; Upadhyaya, Arpita; Tb, Usdin; Bao, Zhejing; Colón-Ramos, Daniel A.; Pl, Riviere; H, Liu; Wu, Yue; Shroff, Hari

doi:10.1101/647370

Cited by 18 publications

(16 citation statements)

References 52 publications

(67 reference statements)

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“…DNNs are versatile tools for various applications, among which image analysis for general purpose feature recognition as well as for optical microscopy are prominent. (22)(23)(24)(25)(26) Recently, the U-net architecture has been demonstrated to be well suited for image segmentation. (27,28) Fundamentally, image segmentation is similar to sBG estimation: A featurethe PSF without BGis overlaid with the sBG, which should be identified from the combined image in order to subsequently remove it.…”

Section: Resultsmentioning

confidence: 99%

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet

Möckl

Roy

Petrov

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3D localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, both for simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of super-resolution reconstructions of biological structures. Significance:A main factor that degrades image quality in fluorescence microscopy is unwanted background fluorescence. Background is almost never uniform, especially in complex samples. Rather, background usually exhibits some structure, making it very difficult to distinguish from the signal of interest. Due to this challenging problem, background fluorescence is often assumed to be uniform even though it is not. This assumption leads to deteriorated image quality, e.g. in localization-based superresolution microscopy, and to the introduction of uncharacterized biases. To overcome this challenge, we developed a general framework rooted in deep learning to accurately and rapidly estimate arbitrarily structured background using several distinct image shapes for the single emitter. Proper background estimation allows for critical performance improvement of various optical microscopy methods. Main Text:

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Section: Resultsmentioning

confidence: 99%

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet

Möckl

Roy

Petrov

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…To visualize the developmental sequence leading to nerve ring assembly, we established an integrated protocol that enables longterm, four-dimensional imaging of living C. elegans embryos, with isotropic spatial resolution 62 . Our system yields minute scale high-resolution 4D data sets, enabling simultaneous tracking of nuclei and neuronal structures [63][64][65][66][67][68] , which can be analyzed to correlate cell-lineage identities with developmental dynamics.…”

Section: Layered Neuropil Architecture Is Influenced By Early Segregamentioning

confidence: 99%

Structural and developmental principles of neuropil assembly inC. elegans

Moyle

Barnes

Kuchroo

et al. 2020

Preprint

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To visualize DC cluster relationships we developed C-PHATE, an extension of PHATE 30 capable of generating a 3D representation of the multigranular structures in the DC hierarchical tree ( Fig. 1a,b; see Methods). Quantitative comparisons of DC/C-PHATE results revealed similar clustering behaviors between the two separately analyzed nervering reconstructions (Adjusted Rand Index (ARI) of 0.7; Extended Data Fig. 1a,b), consistent with the long-accepted qualitative descriptions of the stereotyped structure for the C. elegans nerve ring 3,31 , and recent analyses of neurite adjacencies 22 . Examination of the cluster patterns through the iterations of DC revealed known cell-cell interactions and behavioral circuits ( Fig. 1b,c; Extended Data Fig. 2a,b; [32][33][34][35][36][37][38][39] ), consistent with behavioral and connectomic studies 10,19,20 . The multigranular outputs of the DC/C-PHATE results enabled understanding of the cell-cell interactions within the context of functional circuits, and the functional circuits within the context of the larger bundles of the neuropil.Comparisons of the modularity score, a measure of cluster robustness 40,41 , was highest when the majority of neurites were grouped into four super-clusters ( Fig. 1b; Extended Data Fig. 1a,b; Supplemental Video 1,2; see Methods). Overlaying our analysis of these four super-clusters (see Methods) onto the anatomy of the nerve ring revealed that they correspond to four distinct and tightly grouped bundles within the greater neuropil, circling the pharynx isthmus and stacked along the anterior-posterior axis of the animal. These clusters are herein called S1, S2, S3 and S4 for Stratum 1 etc. (Fig. 1c; Extended Data Fig. 1c-h; Supplemental Video 3 show details of these structures and single-cell identities). The stratified organization of the nerve ring neuropil, resolved here at single neurite level, is reminiscent of laminar organizations in the Drosophila nervous system 42,43 , and in the vertebrate retina and the brain cortex 44,45 .We observed that not all computationally clustered neurites followed simple bundled paths through the neuropil. In Strata 1 (S1), 32 anterior sensory neurons project perpendicular to the nerve ring bundle before making a 180º curl and returning to the anterior limits of the neuropil, where they terminate as synaptic endplates (Fig. 1d; Extended Data Fig. 3a-d; 3,46 ). These uniquely shaped S1 neurons looped at the borders of the S2/S3/S4 bundles. The anterior loops encase ~90% of S2, and the posterior loops encase ~84% of S3 and 100% of S4 ( Fig. 1d-g; Extended Data Fig. 3e-k ; SupplementalVideo 5; Supplemental Table 1). We found that the 32 axons form a six-fold symmetrical honeycomb structure along the neuropil's arc, creating a scaffold that encapsulates the different strata ( Fig. 1g; Extended Data Fig. 3e-h; Supplemental Video 4).The architecture of the neuropil reflects functional segregation of distinct sensory information streams and motor outputs.C. elegans neurons can be divided into three categories ...

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“…For long-term timelapse, Tg(clbnb:lyn-gfp);TgBAC(cxcr4b:h2a-mcherry) double transgenic embryos at 32hpf were embedded in 1% agarose and imaged on a custom DiSPIM 33 using a 40X objective after removal of overlying agarose. Image registration and joint deconvolution was performed using a recently improved pipeline that offers greatly increased processing speed 34 . Registration was performed using the methods in Guo et al , and for deconvolution we used 10 iterations of conventional Richardson-Lucy.…”

Section: Time-lapse Microscopy Segmentation and Quantificationmentioning

confidence: 99%

A sheath of motile cells supports collective migration in of the Zebrafish posterior lateral line primordium under the skin

Nogare

Natesh

Chitnis

2019

Preprint

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During embryonic development, cells must navigate through diverse three-dimensional environments robustly and reproducibly. The zebrafish posterior lateral line primordium (PLLp), a group of approximately 120 cells which migrates from the otic vesicle to the tip of the tail, spearheading the development of the lateral line sensory system, is an excellent model to study such collective migration in an in vivo context. This system migrates in a channel formed by the underlying horizontal myoseptum and somites, and the overlying skin. While cells in the leading part of the PLLp are flat and have a more mesenchymal morphology, cells in the trailing part progressively reorganize to form epithelial rosettes, called protoneuromasts. These epithelial cells extend basal cryptic lamellipodia in the direction of migration in response to both chemokine and FGF signals. In this study, we show that, in addition to these cryptic lamellipodia, the core epithelial cells are in fact surrounded by a population of motile cells which extend actin-rich migratory processes apposed to the overlying skin. These thin cells wrap around the protoneuromasts, forming a continuous sheath of cells around the apical and lateral surface of the PLLp. The processes extended by these cells are highly polarized in the direction of migration and this directionality, like that of the basal lamellipodia, is dependent on FGF signaling. Consistent with interactions of sheath cells with the overlying skin contributing to migration, removal of the skin stalls migration. However, this is accompanied by some surprising changes. There is a profound change in the morphology of the sheath cells, with directional superficial lamellipodia being replaced with the appearance of undirected blebs or ruffles. Furthermore, removal of the skin not only affects underlying lamellipodia, it simultaneously alters the morphology and behavior of the deeper basal cryptic lamellipodia, even though these cells do not directly contact the skin. Directional actin-rich protrusions on both the apical and basal surface and migration are completely and simultaneously restored upon regrowth of the skin over the PLLp. We suggest that this system utilizes a circumferential sheath of motile cells to allow the internal epithelial cells to migrate collectively in the confined space of the horizontal myopseptum and that elastic confinement provided by the overlying skin is essential for effective collective migratory behavior of primordium cells.

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Accelerating iterative deconvolution and multiview fusion by orders of magnitude

Cited by 18 publications

References 52 publications

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet

Accurate and rapid background estimation in single-molecule localization microscopy using the deep neural network BGnet

Structural and developmental principles of neuropil assembly inC. elegans

A sheath of motile cells supports collective migration in of the Zebrafish posterior lateral line primordium under the skin

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