u2os crispr nup96 snap cell line clone30  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH u2os crispr nup96 snap cell line clone30
    U2os Crispr Nup96 Snap Cell Line Clone30, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    u2os crispr nup96 snap cell line clone30  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH u2os crispr nup96 snap cell line clone30
    U2os Crispr Nup96 Snap Cell Line Clone30, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nup96 snap tag cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH nup96 snap tag cells
    (a) A subregion of the reconstructed <t>Nup96</t> using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).
    Nup96 Snap Tag Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Universal inverse modelling of point spread functions for SMLM localization and microscope characterization"

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    Journal: bioRxiv

    doi: 10.1101/2023.10.26.564064

    (a) A subregion of the reconstructed Nup96 using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).
    Figure Legend Snippet: (a) A subregion of the reconstructed Nup96 using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).

    Techniques Used: In Situ, Imaging

    PSF models are estimated from bead data. (a, b) Estimated PSF model and the transformation for a dual-color system. (c, d) 3D dual-color SMLM imaging of NPC, where Nup96-SNAP is labeled with Alexa Fluor 647 and WGA is labeled with CF680. (e) Estimated IAB model for each channel of a 4Pi-SMLM system. (f) Example beads and corresponding forward models at various z and φ positions. (g) Estimated transformation of each target channel to the reference channel of a 4Pi-SMLM system. (h,i) 4Pi-SMLM imaging of Nup96-mMaple. Scale bars, 5 µm (b, g), 2 µm (h), 1 µm (a, c, e, f), 100 nm (d, i).
    Figure Legend Snippet: PSF models are estimated from bead data. (a, b) Estimated PSF model and the transformation for a dual-color system. (c, d) 3D dual-color SMLM imaging of NPC, where Nup96-SNAP is labeled with Alexa Fluor 647 and WGA is labeled with CF680. (e) Estimated IAB model for each channel of a 4Pi-SMLM system. (f) Example beads and corresponding forward models at various z and φ positions. (g) Estimated transformation of each target channel to the reference channel of a 4Pi-SMLM system. (h,i) 4Pi-SMLM imaging of Nup96-mMaple. Scale bars, 5 µm (b, g), 2 µm (h), 1 µm (a, c, e, f), 100 nm (d, i).

    Techniques Used: Transformation Assay, Imaging, Labeling

    (a) uiPSF was used to determine a PSF model from single blinking fluorophores when specific Zernike aberrations were applied by a deformable mirror (DM) with a magnitude of − λ /2 π . (b) The estimated Zernike coefficients from single blinking fluorophores for 25 different Zernike aberrations applied by the DM. Samples for a,b are microtubules labeled with AF647. (c) Tetrapod PSF model generated from a phase plate, the pupil-image based PSF model is estimated from single blinking fluorophores. The sample is TOMM20 labeled with AF647. (d-f) Estimation of a Zernike-based 4Pi-PSF model (estimated PSF model (d) pupil function (e) and Zernike coefficients (f)) from blinking single fluorophores. The sample is Nup96-mMaple in U2OS cells. (g-i) Comparison of PSF models from SMLM data and from bead data for astigmatic 3D SMLM (h). (j) The sample is Nup96-AF647 imaged at 5 µm above the coverslip using an oil immersion objective. (i) XZ view of the selected region in (g), a few zoom-in NPCs under the white arrows shows consistent ring separation for the in-situ PSF model. Scale bars, 1 µm (a,c,d,h,i), 5 µm (g), 200 nm (i, zoom in).
    Figure Legend Snippet: (a) uiPSF was used to determine a PSF model from single blinking fluorophores when specific Zernike aberrations were applied by a deformable mirror (DM) with a magnitude of − λ /2 π . (b) The estimated Zernike coefficients from single blinking fluorophores for 25 different Zernike aberrations applied by the DM. Samples for a,b are microtubules labeled with AF647. (c) Tetrapod PSF model generated from a phase plate, the pupil-image based PSF model is estimated from single blinking fluorophores. The sample is TOMM20 labeled with AF647. (d-f) Estimation of a Zernike-based 4Pi-PSF model (estimated PSF model (d) pupil function (e) and Zernike coefficients (f)) from blinking single fluorophores. The sample is Nup96-mMaple in U2OS cells. (g-i) Comparison of PSF models from SMLM data and from bead data for astigmatic 3D SMLM (h). (j) The sample is Nup96-AF647 imaged at 5 µm above the coverslip using an oil immersion objective. (i) XZ view of the selected region in (g), a few zoom-in NPCs under the white arrows shows consistent ring separation for the in-situ PSF model. Scale bars, 1 µm (a,c,d,h,i), 5 µm (g), 200 nm (i, zoom in).

    Techniques Used: Labeling, Generated, Comparison, In Situ

    Imaging of Nup96-SNAP-AF647 in U2OS cells was performed using an oil immersion objective lens (NA=1.5) on a single-channel SMLM system equipped with a DM. (a) A sandwiched sample of U2OS cells was prepared for an imaging depth of 25 µm. (b) Top view of the reconstructed Nup96 using the in situ PSF model. (c) Enlarged XY view of the selected region in (b). (d) XZ view of the selected region in (c) reconstructed from bead and in situ PSF models, respectively. Scale bars, 2 µm (b), 1 µm (c), and 0.5µm (d).
    Figure Legend Snippet: Imaging of Nup96-SNAP-AF647 in U2OS cells was performed using an oil immersion objective lens (NA=1.5) on a single-channel SMLM system equipped with a DM. (a) A sandwiched sample of U2OS cells was prepared for an imaging depth of 25 µm. (b) Top view of the reconstructed Nup96 using the in situ PSF model. (c) Enlarged XY view of the selected region in (b). (d) XZ view of the selected region in (c) reconstructed from bead and in situ PSF models, respectively. Scale bars, 2 µm (b), 1 µm (c), and 0.5µm (d).

    Techniques Used: Imaging, In Situ

    (a) FD map of Zernike coefficients obtained from beads and single fluorophores in an FOV of about 180 µm × 180 µm. (b) uiPSF successfully recovers FD PSFs from beads at different positions indicated in (a). (c) Comparison of localization bias in x, y, and z obtained through the localization of beads data using the FD PSF and an average PSF. (d-l) in-situ FD PSF. (d) Nup96-AF647 in U2OS cells imaged, fitted with FD-DeepLoc using a FD in situ PSF model. The different colors represent various z positions. (e) and (f) zooms as indicated in (d). e(i-iii) and f(i-iii) are side view reconstructions of the selected region indicated in (e-f) and intensity profiles through individual NPCs (h-l) demonstrate the higher quality for the FD in situ PSF. Scale bars, 50 µm (a), 1 µm (b), 20 µm (d), and 0.5 µm (e-f).
    Figure Legend Snippet: (a) FD map of Zernike coefficients obtained from beads and single fluorophores in an FOV of about 180 µm × 180 µm. (b) uiPSF successfully recovers FD PSFs from beads at different positions indicated in (a). (c) Comparison of localization bias in x, y, and z obtained through the localization of beads data using the FD PSF and an average PSF. (d-l) in-situ FD PSF. (d) Nup96-AF647 in U2OS cells imaged, fitted with FD-DeepLoc using a FD in situ PSF model. The different colors represent various z positions. (e) and (f) zooms as indicated in (d). e(i-iii) and f(i-iii) are side view reconstructions of the selected region indicated in (e-f) and intensity profiles through individual NPCs (h-l) demonstrate the higher quality for the FD in situ PSF. Scale bars, 50 µm (a), 1 µm (b), 20 µm (d), and 0.5 µm (e-f).

    Techniques Used: Comparison, In Situ

    (a) Top view of the reconstructed Nup96 using the in situ PSF model with and without uiPSF guided aberration correction by AO. (b) Zernike aberrations calculated from single molecule data using uiPSF with and without AO correction. (c-d) XZ view of the selected region in (a) for samples with and without AO correction. After uiPSF guided aberration correction, the reconstructed image quality improved significantly as demonstrated by the resolved double ring structure. Scale bars, 1 µm (a), 500 nm (c-d).
    Figure Legend Snippet: (a) Top view of the reconstructed Nup96 using the in situ PSF model with and without uiPSF guided aberration correction by AO. (b) Zernike aberrations calculated from single molecule data using uiPSF with and without AO correction. (c-d) XZ view of the selected region in (a) for samples with and without AO correction. After uiPSF guided aberration correction, the reconstructed image quality improved significantly as demonstrated by the resolved double ring structure. Scale bars, 1 µm (a), 500 nm (c-d).

    Techniques Used: In Situ

    nup96  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH nup96
    Nup96, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nup96  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH nup96
    A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP‐tagged <t>Nup96.</t> Cells were semipermeabilized with digitonin and Nup96 was labeled with the BG‐AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring‐shaped structures built by Y‐complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of four biological replicates. Inset scale bars, 200 nm. Source data are available online for this figure.
    Nup96, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Super‐resolution microscopy reveals focal organization of ER ‐associated Y‐complexes in mitosis"

    Article Title: Super‐resolution microscopy reveals focal organization of ER ‐associated Y‐complexes in mitosis

    Journal: EMBO Reports

    doi: 10.15252/embr.202356766

    A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP‐tagged Nup96. Cells were semipermeabilized with digitonin and Nup96 was labeled with the BG‐AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring‐shaped structures built by Y‐complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of four biological replicates. Inset scale bars, 200 nm. Source data are available online for this figure.
    Figure Legend Snippet: A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP‐tagged Nup96. Cells were semipermeabilized with digitonin and Nup96 was labeled with the BG‐AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring‐shaped structures built by Y‐complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of four biological replicates. Inset scale bars, 200 nm. Source data are available online for this figure.

    Techniques Used: Expressing, Labeling

    A, B 3D dSTORM images of interphase (A) or metaphase‐arrested (B) U2OS cells expressing SNAP‐tagged Nup96 that were semipermeabilized with digitonin and labeled with the BG‐AF647 SNAP dye. The SMLM data were rendered with SMAP. Left panels in (A) and (B) were rendered in a z range of −500 to 500 nm. For the insets 1 and 2, the top panels represent the xy dimension, whereas the bottom panels represent xz dimension. For (A), Y‐rings localized at both faces of interphase NPCs were indicated as R1 and R2. In (B), mitotic clusters at the ER and at kinetochores are magnified in insets 1 and 2, respectively. Localizations were color‐coded according to their z position. The insets were rendered in the z ranges of −100 to 300 nm and −200 to 200 nm for (A) and (B), respectively. Representative images of four biological replicates. Scale bar of left panels, 10 μm. Inset scale bars, 100 nm.
    Figure Legend Snippet: A, B 3D dSTORM images of interphase (A) or metaphase‐arrested (B) U2OS cells expressing SNAP‐tagged Nup96 that were semipermeabilized with digitonin and labeled with the BG‐AF647 SNAP dye. The SMLM data were rendered with SMAP. Left panels in (A) and (B) were rendered in a z range of −500 to 500 nm. For the insets 1 and 2, the top panels represent the xy dimension, whereas the bottom panels represent xz dimension. For (A), Y‐rings localized at both faces of interphase NPCs were indicated as R1 and R2. In (B), mitotic clusters at the ER and at kinetochores are magnified in insets 1 and 2, respectively. Localizations were color‐coded according to their z position. The insets were rendered in the z ranges of −100 to 300 nm and −200 to 200 nm for (A) and (B), respectively. Representative images of four biological replicates. Scale bar of left panels, 10 μm. Inset scale bars, 100 nm.

    Techniques Used: Expressing, Labeling

    2D dSTORM images of a metaphase‐arrested U2OS cell expressing Nup96 with a SNAP‐tag, labeled with BG‐Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho‐histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data were reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm. Data analysis procedure of SMLM data. SMLM data were reconstructed in SMAP, processed using DBSCAN and then filtered to obtain the geometry and stoichiometry of Y‐complexes in interphase NPCs, mitotic clusters and at kinetochores. Quantification of the number of localizations in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Quantification of the number of protein copies in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Quantification of the cluster width of interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Source data are available online for this figure.
    Figure Legend Snippet: 2D dSTORM images of a metaphase‐arrested U2OS cell expressing Nup96 with a SNAP‐tag, labeled with BG‐Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho‐histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data were reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm. Data analysis procedure of SMLM data. SMLM data were reconstructed in SMAP, processed using DBSCAN and then filtered to obtain the geometry and stoichiometry of Y‐complexes in interphase NPCs, mitotic clusters and at kinetochores. Quantification of the number of localizations in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Quantification of the number of protein copies in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Quantification of the cluster width of interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. Source data are available online for this figure.

    Techniques Used: Expressing, Labeling, Imaging

    U2OS cells expressing Nup96‐SNAP were arrested at the metaphase‐anaphase transition using cell synchronization and treatment with 20 μM MG132, as described in Fig . Then, they were fixed, labeled with the BG‐AF647 SNAP dye, and immunostained with antibodies targeting CREST and Climp63 to label kinetochores and the ER, respectively. DNA was visualized with Hoechst (visible only in merged channels). N = 3, n = 30. Scale bar, 5 μm and inset scale bar, 1 μm.
    Figure Legend Snippet: U2OS cells expressing Nup96‐SNAP were arrested at the metaphase‐anaphase transition using cell synchronization and treatment with 20 μM MG132, as described in Fig . Then, they were fixed, labeled with the BG‐AF647 SNAP dye, and immunostained with antibodies targeting CREST and Climp63 to label kinetochores and the ER, respectively. DNA was visualized with Hoechst (visible only in merged channels). N = 3, n = 30. Scale bar, 5 μm and inset scale bar, 1 μm.

    Techniques Used: Expressing, Labeling

    cell lines cell line source s u2os nup96 snap tag  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH cell lines cell line source s u2os nup96 snap tag
    Cell Lines Cell Line Source S U2os Nup96 Snap Tag, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH nup96 snap tag cells
    a–f , Nuclear pore complexes. a , Representative image of <t>Nup96-labeled</t> NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , Single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetry model (cyan) shown in c . The model is parameterized by the listed parameters (blue). d–f , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.6 nm ( e ) and twist θ = 8.8 ± 9.0° ( f ). Sim, simulated data (gray, see also Extended Data Fig. ). Simulation parameters are summarized in Supplementary Table . Sample size: sites, n s = 3,517; cells, n c = 5. xy denotes the top view and xz the side view in all parts of the figure. g – l , Microtubules. g , Representative image of immunolabeled microtubules in a 3D dataset (top view; original data from Speiser et al. ). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 23.8 ± 1.5 nm, based on segments of 1 µm length. Sample size n s = 161, n c = 1. k , Top view of a region containing a 5.2-µm-long non-overlapping (boxed) curved segment ( l ). l , The long segment without (left) and with (right) the fitted model overlaid. Insets are the cross-sections of the boxed short segments. Reported values are mean ± s.d., based on n s sites in a total of n c cells. Scale bars: a , g , k , l , 1 μm; b , h , 100 nm; insets in l , 50 nm.
    Nup96 Snap Tag Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Maximum-likelihood model fitting for quantitative analysis of SMLM data"

    Article Title: Maximum-likelihood model fitting for quantitative analysis of SMLM data

    Journal: Nature Methods

    doi: 10.1038/s41592-022-01676-z

    a–f , Nuclear pore complexes. a , Representative image of Nup96-labeled NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , Single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetry model (cyan) shown in c . The model is parameterized by the listed parameters (blue). d–f , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.6 nm ( e ) and twist θ = 8.8 ± 9.0° ( f ). Sim, simulated data (gray, see also Extended Data Fig. ). Simulation parameters are summarized in Supplementary Table . Sample size: sites, n s = 3,517; cells, n c = 5. xy denotes the top view and xz the side view in all parts of the figure. g – l , Microtubules. g , Representative image of immunolabeled microtubules in a 3D dataset (top view; original data from Speiser et al. ). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 23.8 ± 1.5 nm, based on segments of 1 µm length. Sample size n s = 161, n c = 1. k , Top view of a region containing a 5.2-µm-long non-overlapping (boxed) curved segment ( l ). l , The long segment without (left) and with (right) the fitted model overlaid. Insets are the cross-sections of the boxed short segments. Reported values are mean ± s.d., based on n s sites in a total of n c cells. Scale bars: a , g , k , l , 1 μm; b , h , 100 nm; insets in l , 50 nm.
    Figure Legend Snippet: a–f , Nuclear pore complexes. a , Representative image of Nup96-labeled NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , Single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetry model (cyan) shown in c . The model is parameterized by the listed parameters (blue). d–f , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.6 nm ( e ) and twist θ = 8.8 ± 9.0° ( f ). Sim, simulated data (gray, see also Extended Data Fig. ). Simulation parameters are summarized in Supplementary Table . Sample size: sites, n s = 3,517; cells, n c = 5. xy denotes the top view and xz the side view in all parts of the figure. g – l , Microtubules. g , Representative image of immunolabeled microtubules in a 3D dataset (top view; original data from Speiser et al. ). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 23.8 ± 1.5 nm, based on segments of 1 µm length. Sample size n s = 161, n c = 1. k , Top view of a region containing a 5.2-µm-long non-overlapping (boxed) curved segment ( l ). l , The long segment without (left) and with (right) the fitted model overlaid. Insets are the cross-sections of the boxed short segments. Reported values are mean ± s.d., based on n s sites in a total of n c cells. Scale bars: a , g , k , l , 1 μm; b , h , 100 nm; insets in l , 50 nm.

    Techniques Used: Labeling, Immunolabeling

    a–e, Simulated NPCs based on experimental parameters. a , Representative images of the simulated Nup96-labeled nuclear pore complexes (NPC) in 3D (top view). b , single NPCs (orange) as indicated in a are fitted with the eight-fold symmetry model. c-e , Histograms of three fitted parameters: radius r = 53.4 ± 1.1 nm ( c ), separation s = 50.0 ± 3.8 nm ( d ), and twist \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta = 8.9 \pm 4.7^\circ$$\end{document} θ = 8.9 ± 4 . 7 ∘ ( e ). Shown values are mean ± s.d. Sample size: n s = 3,539. f–h, Runtime estimates based on the simulated microtubule segments with increasing arc lengths . The segments were derived from the same simulated long sinusoidal microtubule. f , The longest segment fitted with the model having the longest arc length (5.2 μm), as the example. g , relation between fitting runtime t and the model arc length n and fit with power model t ~ n α resulting in α = 3.1. h , Measured radii r against different segment lengths. The horizontal line indicates the ground truth. Sample size: n s = 1. Simulation parameters are listed in Supplementary Table . Scale bars: 100 nm ( a , b ), 500 nm ( f ).
    Figure Legend Snippet: a–e, Simulated NPCs based on experimental parameters. a , Representative images of the simulated Nup96-labeled nuclear pore complexes (NPC) in 3D (top view). b , single NPCs (orange) as indicated in a are fitted with the eight-fold symmetry model. c-e , Histograms of three fitted parameters: radius r = 53.4 ± 1.1 nm ( c ), separation s = 50.0 ± 3.8 nm ( d ), and twist \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta = 8.9 \pm 4.7^\circ$$\end{document} θ = 8.9 ± 4 . 7 ∘ ( e ). Shown values are mean ± s.d. Sample size: n s = 3,539. f–h, Runtime estimates based on the simulated microtubule segments with increasing arc lengths . The segments were derived from the same simulated long sinusoidal microtubule. f , The longest segment fitted with the model having the longest arc length (5.2 μm), as the example. g , relation between fitting runtime t and the model arc length n and fit with power model t ~ n α resulting in α = 3.1. h , Measured radii r against different segment lengths. The horizontal line indicates the ground truth. Sample size: n s = 1. Simulation parameters are listed in Supplementary Table . Scale bars: 100 nm ( a , b ), 500 nm ( f ).

    Techniques Used: Labeling, Derivative Assay

    a–d , The nuclear pore complex. a , b , Representative images of individual sites showing Nup96-SNAP-AF647 and immunolabeled Elys-CF680 ( a ) or Nup133-CF680 ( b ) . c , A model fit to the reference protein Nup96 enables the registration of all sites of one dataset and integration of the different dual-color datasets into one common coordinate system ( d ). CR, cytoplasmic ring; NR, nucleoplasmic ring. See Supplementary Video . Sample size: sites: Elys, n s = 1,875; Nup133, n s = 1,739; Nup62, n s = 2,263; Nup153, n s = 2,159; WGA, n s = 1,778; cells: n c = 3 for all. e–i , Dynamic dual-color reconstruction of endocytosis in yeast. e , Overview image of a single yeast cell showing Abp1-mMaple and Las17-SNAP-AF647. f , Individual endocytic sites are fitted with a dual-color model ( h ) that reflects the expected distribution ( g ) of Abp1 and Las17: we model Abp1 as a hemiellipsoid and Las17 as a thick ring and project these geometries in 2D. The fitted axial length of Abp1 is used as a proxy for pseudotime to sort individual endocytic sites according to their progression along the endocytic timeline. The fitted position and orientation are then used to average all sites in each time bin ( i ). Bin size: 21 sites. Sample size: n s = 130, n c = 51. A running average is shown in Supplementary Video . Scale bars: a – d , f , i , 100 nm; e , 500 μm.
    Figure Legend Snippet: a–d , The nuclear pore complex. a , b , Representative images of individual sites showing Nup96-SNAP-AF647 and immunolabeled Elys-CF680 ( a ) or Nup133-CF680 ( b ) . c , A model fit to the reference protein Nup96 enables the registration of all sites of one dataset and integration of the different dual-color datasets into one common coordinate system ( d ). CR, cytoplasmic ring; NR, nucleoplasmic ring. See Supplementary Video . Sample size: sites: Elys, n s = 1,875; Nup133, n s = 1,739; Nup62, n s = 2,263; Nup153, n s = 2,159; WGA, n s = 1,778; cells: n c = 3 for all. e–i , Dynamic dual-color reconstruction of endocytosis in yeast. e , Overview image of a single yeast cell showing Abp1-mMaple and Las17-SNAP-AF647. f , Individual endocytic sites are fitted with a dual-color model ( h ) that reflects the expected distribution ( g ) of Abp1 and Las17: we model Abp1 as a hemiellipsoid and Las17 as a thick ring and project these geometries in 2D. The fitted axial length of Abp1 is used as a proxy for pseudotime to sort individual endocytic sites according to their progression along the endocytic timeline. The fitted position and orientation are then used to average all sites in each time bin ( i ). Bin size: 21 sites. Sample size: n s = 130, n c = 51. A running average is shown in Supplementary Video . Scale bars: a – d , f , i , 100 nm; e , 500 μm.

    Techniques Used: Immunolabeling

    a–e , Workflow. a , Example NPC particles. We assumed that all sites are samples of the same underlying distribution. b , All-versus-all comparison. We first determine the site (in the example, the site ii) that best describes all of the other sites based on the rank on sum LL of the all-to-all matrix, where the 50 subset sites were fitted to each other. c , Construction of the initial template. The initial data-driven template is built based on sequential registration in the order of the sum LL rank. d , Iterative registration. The final fused particle is used to register all sites in the 1,312-site dataset. This procedure yields an updated fused particle, which is used to register the dataset again. This process is iterated until it converges ( e ). f–h , The final average calculated from 1,312 particles without any assumption on the underlying geometry or symmetry in a tilted view ( f ), and for comparison the electron microscopy density (PDB ID: 5A9Q ) of the NPC with the C termini of Nup96 indicated in red ( g , adapted with permission from ref. , Springer Nature). h , Top and side view, where the nucleoplasmic and cytoplasmic rings are shown together (left panel), or separately (middle, right panels). The two proteins per ring per symmetry unit give rise to tilted elongated average protein distributions in the averages (arrows in h ). See Supplementary Video . Scale bars: 50 nm.
    Figure Legend Snippet: a–e , Workflow. a , Example NPC particles. We assumed that all sites are samples of the same underlying distribution. b , All-versus-all comparison. We first determine the site (in the example, the site ii) that best describes all of the other sites based on the rank on sum LL of the all-to-all matrix, where the 50 subset sites were fitted to each other. c , Construction of the initial template. The initial data-driven template is built based on sequential registration in the order of the sum LL rank. d , Iterative registration. The final fused particle is used to register all sites in the 1,312-site dataset. This procedure yields an updated fused particle, which is used to register the dataset again. This process is iterated until it converges ( e ). f–h , The final average calculated from 1,312 particles without any assumption on the underlying geometry or symmetry in a tilted view ( f ), and for comparison the electron microscopy density (PDB ID: 5A9Q ) of the NPC with the C termini of Nup96 indicated in red ( g , adapted with permission from ref. , Springer Nature). h , Top and side view, where the nucleoplasmic and cytoplasmic rings are shown together (left panel), or separately (middle, right panels). The two proteins per ring per symmetry unit give rise to tilted elongated average protein distributions in the averages (arrows in h ). See Supplementary Video . Scale bars: 50 nm.

    Techniques Used: Electron Microscopy

    u 2 os crispr nup96 snap  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH u 2 os crispr nup96 snap
    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed <t>U2OS-Nup96-SNAP</t> cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.
    U 2 Os Crispr Nup96 Snap, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The miEye: Bench-top super-resolution microscope with cost-effective equipment"

    Article Title: The miEye: Bench-top super-resolution microscope with cost-effective equipment

    Journal: HardwareX

    doi: 10.1016/j.ohx.2022.e00368

    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed U2OS-Nup96-SNAP cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.
    Figure Legend Snippet: dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed U2OS-Nup96-SNAP cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.

    Techniques Used: Imaging, Labeling

    u-2 os-crispr-nup96-snap clone no.33  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH u-2 os-crispr-nup96-snap clone no.33
    U 2 Os Crispr Nup96 Snap Clone No.33, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nup96 snap tag cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH nup96 snap tag cells
    a-f , Quantification of NPCs . a , Representative image of the <t>Nup96-labeled</t> NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetric model (cyan) shown in ( c ). The model is parameterized by the listed parameters (blue). d-e , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.8 nm ( e ), and twist θ = 8.8 ± 9.0° ( f ). ‘Sim’ stands for simulation (gray). Sample size: n s = 3,524, n c = 5. g-j , Quantification of microtubules . g , Representative image of immunolabeled microtubules in a 3D dataset (top view). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 24.1 ± 3.4 nm, based on segments of 1 μm length. Sample size n s = 161, n c = 1. Shown values are mean± s.d., based on n s sites in total n c cells. Scale bars, 1 μm ( a,g ), 100 nm ( b,h ).
    Nup96 Snap Tag Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Maximum-likelihood model fitting for quantitative analysis of SMLM data"

    Article Title: Maximum-likelihood model fitting for quantitative analysis of SMLM data

    Journal: bioRxiv

    doi: 10.1101/2021.08.30.456756

    a-f , Quantification of NPCs . a , Representative image of the Nup96-labeled NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetric model (cyan) shown in ( c ). The model is parameterized by the listed parameters (blue). d-e , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.8 nm ( e ), and twist θ = 8.8 ± 9.0° ( f ). ‘Sim’ stands for simulation (gray). Sample size: n s = 3,524, n c = 5. g-j , Quantification of microtubules . g , Representative image of immunolabeled microtubules in a 3D dataset (top view). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 24.1 ± 3.4 nm, based on segments of 1 μm length. Sample size n s = 161, n c = 1. Shown values are mean± s.d., based on n s sites in total n c cells. Scale bars, 1 μm ( a,g ), 100 nm ( b,h ).
    Figure Legend Snippet: a-f , Quantification of NPCs . a , Representative image of the Nup96-labeled NPCs (Nup96-SNAP-AF647) in a 3D dataset (top view). b , single NPCs (localizations in orange) as indicated in a are fitted with the eight-fold symmetric model (cyan) shown in ( c ). The model is parameterized by the listed parameters (blue). d-e , Histograms of three fitted parameters: radius r = 53.4 ± 2.3 nm ( d ), separation s = 50.2 ± 5.8 nm ( e ), and twist θ = 8.8 ± 9.0° ( f ). ‘Sim’ stands for simulation (gray). Sample size: n s = 3,524, n c = 5. g-j , Quantification of microtubules . g , Representative image of immunolabeled microtubules in a 3D dataset (top view). h , One microtubule segment (red) as indicated in g is fitted by the linear-tube model ( i ). The fitted model is indicated in cyan. i , The linear-tube model parameterized by the listed parameters (blue), the control points c i define a cubic spline. j , Histogram of the fitted radius r = 24.1 ± 3.4 nm, based on segments of 1 μm length. Sample size n s = 161, n c = 1. Shown values are mean± s.d., based on n s sites in total n c cells. Scale bars, 1 μm ( a,g ), 100 nm ( b,h ).

    Techniques Used: Labeling, Immunolabeling

    a , Representative images of the simulated Nup96-labeled NPCs in 3D (top view). b , single NPCs (orange) as indicated in a are fitted with the eight-fold symmetric model. c-e , Histograms of three fitted parameters: radius r = 53.5 ± 1.5 nm ( c ), separation s = 50.3 ± 3.8 nm ( d ), and twist θ = 8.6 ± 5.9° ( e ). Simulation parameters are summarized in . Sample size: n s = 4,000. Scale bars: 100 nm.
    Figure Legend Snippet: a , Representative images of the simulated Nup96-labeled NPCs in 3D (top view). b , single NPCs (orange) as indicated in a are fitted with the eight-fold symmetric model. c-e , Histograms of three fitted parameters: radius r = 53.5 ± 1.5 nm ( c ), separation s = 50.3 ± 3.8 nm ( d ), and twist θ = 8.6 ± 5.9° ( e ). Simulation parameters are summarized in . Sample size: n s = 4,000. Scale bars: 100 nm.

    Techniques Used: Labeling

    a-d, Average density map of the nuclear pore complex. a-b, representative images of individual sites showing Nup96-SNAP-AF647 and immunolabeled Elys-CF680 ( a ) or Nup133-CF680 ( b ). c, A model fit to the reference protein Nup96 allows registering all sites of one data set and integrating different dual-color data sets into one common coordinate system ( d ). NR and CR denotes nucleoplasmic and cytoplasmic rings, respectively. See Supplementary Movie 1. Sample size: Elys: n s = 704; Nup133: n s = 704; Nup62: n s = 605; Nup153: n s = 419; WGA: n s = 488. n c = 1 for all. e-i, Dual-color dynamic reconstruction of endocytosis in yeast. e, Overview image of a single yeast cell showing Abp1-mMaple and Las17-SNAP-AF647. f , Individual endocytic sites are fitted with a dual-color model ( h ) that reflects the expected distribution ( g ) of Abp1 and Las17 : We model Abp1 as a semi-ellipsoid and Las17 as a thick ring and project these geometries in 2D. The fitted axial length of Abp1 is used as a proxy for pseudotime to sort individual endocytic sites according to their progression along the endocytic time line. The fitted position and orientation are then used to average all sites in each time bin ( i ). Bin size: 21 sites. Sample size: n s = 130, n c = 51. A running average can be found in Supplementary Movie 2. Scale bars: 100 nm ( a-d,f,i ), 500 μm ( e ).
    Figure Legend Snippet: a-d, Average density map of the nuclear pore complex. a-b, representative images of individual sites showing Nup96-SNAP-AF647 and immunolabeled Elys-CF680 ( a ) or Nup133-CF680 ( b ). c, A model fit to the reference protein Nup96 allows registering all sites of one data set and integrating different dual-color data sets into one common coordinate system ( d ). NR and CR denotes nucleoplasmic and cytoplasmic rings, respectively. See Supplementary Movie 1. Sample size: Elys: n s = 704; Nup133: n s = 704; Nup62: n s = 605; Nup153: n s = 419; WGA: n s = 488. n c = 1 for all. e-i, Dual-color dynamic reconstruction of endocytosis in yeast. e, Overview image of a single yeast cell showing Abp1-mMaple and Las17-SNAP-AF647. f , Individual endocytic sites are fitted with a dual-color model ( h ) that reflects the expected distribution ( g ) of Abp1 and Las17 : We model Abp1 as a semi-ellipsoid and Las17 as a thick ring and project these geometries in 2D. The fitted axial length of Abp1 is used as a proxy for pseudotime to sort individual endocytic sites according to their progression along the endocytic time line. The fitted position and orientation are then used to average all sites in each time bin ( i ). Bin size: 21 sites. Sample size: n s = 130, n c = 51. A running average can be found in Supplementary Movie 2. Scale bars: 100 nm ( a-d,f,i ), 500 μm ( e ).

    Techniques Used: Immunolabeling

    a-e, Workflow. a , Example single NPC particles. We assumed that all the sites are samples of the same underlying distribution. b , We first find a site (in the example the site iv) that best describes all other sites based on the rank on sum log-likelihood (LL) of the all-to-all matrix, where the 50 subset sites were fitted to each other. c , The initial template is built based on sequential registration in the order of the sum LL rank. d , The final fused particle is used to register all sites in the 318-site data set. This procedure yields an updated fused particle, which is used to register the data set again. This process is iterated until convergence. f-h , The final average calculated from 318 particles without any assumption on the underlying geometry or symmetry in a tilted view ( f ), and for comparison the EM density of the NPC with C-termini of Nup96 indicated in red. ( g ), Top and side view, where the nucleoplasmic and cytoplasmic rings are shown together (left panel), or separately (middle, right panels) ( h ). See Supplementary Movie 3. The two proteins per ring per symmetric unit give rise to tilted elongated average protein distributions in the averages (arrows in h ). Scale bars: 50 nm.
    Figure Legend Snippet: a-e, Workflow. a , Example single NPC particles. We assumed that all the sites are samples of the same underlying distribution. b , We first find a site (in the example the site iv) that best describes all other sites based on the rank on sum log-likelihood (LL) of the all-to-all matrix, where the 50 subset sites were fitted to each other. c , The initial template is built based on sequential registration in the order of the sum LL rank. d , The final fused particle is used to register all sites in the 318-site data set. This procedure yields an updated fused particle, which is used to register the data set again. This process is iterated until convergence. f-h , The final average calculated from 318 particles without any assumption on the underlying geometry or symmetry in a tilted view ( f ), and for comparison the EM density of the NPC with C-termini of Nup96 indicated in red. ( g ), Top and side view, where the nucleoplasmic and cytoplasmic rings are shown together (left panel), or separately (middle, right panels) ( h ). See Supplementary Movie 3. The two proteins per ring per symmetric unit give rise to tilted elongated average protein distributions in the averages (arrows in h ). Scale bars: 50 nm.

    Techniques Used:


    Figure Legend Snippet:

    Techniques Used:


    Figure Legend Snippet:

    Techniques Used:

    2x coverslips  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH 2x coverslips
    2x Coverslips, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH u2os crispr nup96 snap cell line clone30
    U2os Crispr Nup96 Snap Cell Line Clone30, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH nup96 snap tag cells
    (a) A subregion of the reconstructed <t>Nup96</t> using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).
    Nup96 Snap Tag Cells, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH nup96
    (a) A subregion of the reconstructed <t>Nup96</t> using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).
    Nup96, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH cell lines cell line source s u2os nup96 snap tag
    (a) A subregion of the reconstructed <t>Nup96</t> using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).
    Cell Lines Cell Line Source S U2os Nup96 Snap Tag, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH u 2 os crispr nup96 snap
    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed <t>U2OS-Nup96-SNAP</t> cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.
    U 2 Os Crispr Nup96 Snap, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u 2 os crispr nup96 snap/product/CLS Cell Lines Service GmbH
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    CLS Cell Lines Service GmbH u-2 os-crispr-nup96-snap clone no.33
    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed <t>U2OS-Nup96-SNAP</t> cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.
    U 2 Os Crispr Nup96 Snap Clone No.33, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH 2x coverslips
    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed <t>U2OS-Nup96-SNAP</t> cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.
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    Image Search Results


    (a) A subregion of the reconstructed Nup96 using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: (a) A subregion of the reconstructed Nup96 using the in situ PSF model. (b-c) XZ view of selected region in a from bead and in situ PSF models. (d) and (e) are the ring distance of Nup96 as a function of z-position at different depth reconstructed by using beads PSF model and in situ PSF model respectively. The points of various colors represent different imaging depths of Nup96. The straight lines represent the linear regression of the axial distance and z position for each imaging depth, with k representing the corresponding slope. An oil immersion objective lens (NA = 1.43) was used for imaging. (f) and (g) have the same meaning as (d) and (e), respectively, but imaged by a silicone oil immersion objective lens (NA = 1.35). Scale bars, 1 µm (a), 500 nm (b).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: In Situ, Imaging

    PSF models are estimated from bead data. (a, b) Estimated PSF model and the transformation for a dual-color system. (c, d) 3D dual-color SMLM imaging of NPC, where Nup96-SNAP is labeled with Alexa Fluor 647 and WGA is labeled with CF680. (e) Estimated IAB model for each channel of a 4Pi-SMLM system. (f) Example beads and corresponding forward models at various z and φ positions. (g) Estimated transformation of each target channel to the reference channel of a 4Pi-SMLM system. (h,i) 4Pi-SMLM imaging of Nup96-mMaple. Scale bars, 5 µm (b, g), 2 µm (h), 1 µm (a, c, e, f), 100 nm (d, i).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: PSF models are estimated from bead data. (a, b) Estimated PSF model and the transformation for a dual-color system. (c, d) 3D dual-color SMLM imaging of NPC, where Nup96-SNAP is labeled with Alexa Fluor 647 and WGA is labeled with CF680. (e) Estimated IAB model for each channel of a 4Pi-SMLM system. (f) Example beads and corresponding forward models at various z and φ positions. (g) Estimated transformation of each target channel to the reference channel of a 4Pi-SMLM system. (h,i) 4Pi-SMLM imaging of Nup96-mMaple. Scale bars, 5 µm (b, g), 2 µm (h), 1 µm (a, c, e, f), 100 nm (d, i).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: Transformation Assay, Imaging, Labeling

    (a) uiPSF was used to determine a PSF model from single blinking fluorophores when specific Zernike aberrations were applied by a deformable mirror (DM) with a magnitude of − λ /2 π . (b) The estimated Zernike coefficients from single blinking fluorophores for 25 different Zernike aberrations applied by the DM. Samples for a,b are microtubules labeled with AF647. (c) Tetrapod PSF model generated from a phase plate, the pupil-image based PSF model is estimated from single blinking fluorophores. The sample is TOMM20 labeled with AF647. (d-f) Estimation of a Zernike-based 4Pi-PSF model (estimated PSF model (d) pupil function (e) and Zernike coefficients (f)) from blinking single fluorophores. The sample is Nup96-mMaple in U2OS cells. (g-i) Comparison of PSF models from SMLM data and from bead data for astigmatic 3D SMLM (h). (j) The sample is Nup96-AF647 imaged at 5 µm above the coverslip using an oil immersion objective. (i) XZ view of the selected region in (g), a few zoom-in NPCs under the white arrows shows consistent ring separation for the in-situ PSF model. Scale bars, 1 µm (a,c,d,h,i), 5 µm (g), 200 nm (i, zoom in).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: (a) uiPSF was used to determine a PSF model from single blinking fluorophores when specific Zernike aberrations were applied by a deformable mirror (DM) with a magnitude of − λ /2 π . (b) The estimated Zernike coefficients from single blinking fluorophores for 25 different Zernike aberrations applied by the DM. Samples for a,b are microtubules labeled with AF647. (c) Tetrapod PSF model generated from a phase plate, the pupil-image based PSF model is estimated from single blinking fluorophores. The sample is TOMM20 labeled with AF647. (d-f) Estimation of a Zernike-based 4Pi-PSF model (estimated PSF model (d) pupil function (e) and Zernike coefficients (f)) from blinking single fluorophores. The sample is Nup96-mMaple in U2OS cells. (g-i) Comparison of PSF models from SMLM data and from bead data for astigmatic 3D SMLM (h). (j) The sample is Nup96-AF647 imaged at 5 µm above the coverslip using an oil immersion objective. (i) XZ view of the selected region in (g), a few zoom-in NPCs under the white arrows shows consistent ring separation for the in-situ PSF model. Scale bars, 1 µm (a,c,d,h,i), 5 µm (g), 200 nm (i, zoom in).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: Labeling, Generated, Comparison, In Situ

    Imaging of Nup96-SNAP-AF647 in U2OS cells was performed using an oil immersion objective lens (NA=1.5) on a single-channel SMLM system equipped with a DM. (a) A sandwiched sample of U2OS cells was prepared for an imaging depth of 25 µm. (b) Top view of the reconstructed Nup96 using the in situ PSF model. (c) Enlarged XY view of the selected region in (b). (d) XZ view of the selected region in (c) reconstructed from bead and in situ PSF models, respectively. Scale bars, 2 µm (b), 1 µm (c), and 0.5µm (d).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: Imaging of Nup96-SNAP-AF647 in U2OS cells was performed using an oil immersion objective lens (NA=1.5) on a single-channel SMLM system equipped with a DM. (a) A sandwiched sample of U2OS cells was prepared for an imaging depth of 25 µm. (b) Top view of the reconstructed Nup96 using the in situ PSF model. (c) Enlarged XY view of the selected region in (b). (d) XZ view of the selected region in (c) reconstructed from bead and in situ PSF models, respectively. Scale bars, 2 µm (b), 1 µm (c), and 0.5µm (d).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: Imaging, In Situ

    (a) FD map of Zernike coefficients obtained from beads and single fluorophores in an FOV of about 180 µm × 180 µm. (b) uiPSF successfully recovers FD PSFs from beads at different positions indicated in (a). (c) Comparison of localization bias in x, y, and z obtained through the localization of beads data using the FD PSF and an average PSF. (d-l) in-situ FD PSF. (d) Nup96-AF647 in U2OS cells imaged, fitted with FD-DeepLoc using a FD in situ PSF model. The different colors represent various z positions. (e) and (f) zooms as indicated in (d). e(i-iii) and f(i-iii) are side view reconstructions of the selected region indicated in (e-f) and intensity profiles through individual NPCs (h-l) demonstrate the higher quality for the FD in situ PSF. Scale bars, 50 µm (a), 1 µm (b), 20 µm (d), and 0.5 µm (e-f).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: (a) FD map of Zernike coefficients obtained from beads and single fluorophores in an FOV of about 180 µm × 180 µm. (b) uiPSF successfully recovers FD PSFs from beads at different positions indicated in (a). (c) Comparison of localization bias in x, y, and z obtained through the localization of beads data using the FD PSF and an average PSF. (d-l) in-situ FD PSF. (d) Nup96-AF647 in U2OS cells imaged, fitted with FD-DeepLoc using a FD in situ PSF model. The different colors represent various z positions. (e) and (f) zooms as indicated in (d). e(i-iii) and f(i-iii) are side view reconstructions of the selected region indicated in (e-f) and intensity profiles through individual NPCs (h-l) demonstrate the higher quality for the FD in situ PSF. Scale bars, 50 µm (a), 1 µm (b), 20 µm (d), and 0.5 µm (e-f).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: Comparison, In Situ

    (a) Top view of the reconstructed Nup96 using the in situ PSF model with and without uiPSF guided aberration correction by AO. (b) Zernike aberrations calculated from single molecule data using uiPSF with and without AO correction. (c-d) XZ view of the selected region in (a) for samples with and without AO correction. After uiPSF guided aberration correction, the reconstructed image quality improved significantly as demonstrated by the resolved double ring structure. Scale bars, 1 µm (a), 500 nm (c-d).

    Journal: bioRxiv

    Article Title: Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

    doi: 10.1101/2023.10.26.564064

    Figure Lengend Snippet: (a) Top view of the reconstructed Nup96 using the in situ PSF model with and without uiPSF guided aberration correction by AO. (b) Zernike aberrations calculated from single molecule data using uiPSF with and without AO correction. (c-d) XZ view of the selected region in (a) for samples with and without AO correction. After uiPSF guided aberration correction, the reconstructed image quality improved significantly as demonstrated by the resolved double ring structure. Scale bars, 1 µm (a), 500 nm (c-d).

    Article Snippet: Nup96-SNAP-tag cells (catalog no. 300444, CLS Cell Line Service) were rinsed twice with warm PBS.

    Techniques: In Situ

    dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed U2OS-Nup96-SNAP cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.

    Journal: HardwareX

    Article Title: The miEye: Bench-top super-resolution microscope with cost-effective equipment

    doi: 10.1016/j.ohx.2022.e00368

    Figure Lengend Snippet: dSTORM imaging of fluorescently labeled nuclear pore complexes (NPCs) of fixed U2OS-Nup96-SNAP cells. A) Averaged image of the unprocessed 20,000 frame-long image stack showing an individual nucleus of a U2OS cell. B) Reconstructed super-resolution image of Nup96-SNAP-AF647-containing NPCs located at the distinct positions of a single U2OS cell’s entire nucleus. C) Close-up view of a chosen nucleus area (indicated by a white box in panel B ). Individual NPCs and their ring-like arrangement are visible. D) Fourier ring correlation (FRC) curve indicating the obtained spatial resolution of 34.7 nm.

    Article Snippet: Preparation of the U-2 OS cells with labeled Nup96 complexes for dSTORM imaging. a) U-2 OS-CRISPR-Nup96-SNAP clone #33 (300444, CLS GmbH) cell line was purchased from Cell Lines Service (Germany).

    Techniques: Imaging, Labeling