synaptotagmin  (Synaptic Systems)


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    Structured Review

    Synaptic Systems synaptotagmin
    Cellular localization of alpha (α)‐amylase in primary mouse neurons. Image in (a) shows immunofluorescent staining of α‐amylase with antibody directed against salivary α‐amylase (green) and neuronal marker microtubule‐associated protein 2 (MAP2) (magenta). The α‐amylase immunoreactivity is seen in the cell body (arrowhead) and along the dendrites (arrow). Confocal image in (b) shows a primary mouse neuronal process where α‐amylase staining (green) and phalloidin (magenta) are in close association. Confocal image in (c) shows immunofluorescent staining of primary mouse neuronal process where α‐amylase (green) and <t>synaptotagmin</t> (magenta) are in close association (arrow). Confocal image in (d) shows a close association between α‐amylase (green) and CAMKII (magenta) (arrow). Confocal image in (e) shows a primary mouse neuron stained against α‐amylase (green) and glycogen (magenta). The white squares indicate magnification of the area seen in (f and g). Arrows in (f‐g) indicate an close association between α‐amylase and glycogen. Scale bar in (a and e) indicates 5 μm, scale bar in (d and g) indicates 0.5 μm
    Synaptotagmin, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 18 article reviews
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    synaptotagmin - by Bioz Stars, 2022-11
    95/100 stars

    Images

    1) Product Images from "Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology). Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology"

    Article Title: Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology). Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology

    Journal: Aging Cell

    doi: 10.1111/acel.13433

    Cellular localization of alpha (α)‐amylase in primary mouse neurons. Image in (a) shows immunofluorescent staining of α‐amylase with antibody directed against salivary α‐amylase (green) and neuronal marker microtubule‐associated protein 2 (MAP2) (magenta). The α‐amylase immunoreactivity is seen in the cell body (arrowhead) and along the dendrites (arrow). Confocal image in (b) shows a primary mouse neuronal process where α‐amylase staining (green) and phalloidin (magenta) are in close association. Confocal image in (c) shows immunofluorescent staining of primary mouse neuronal process where α‐amylase (green) and synaptotagmin (magenta) are in close association (arrow). Confocal image in (d) shows a close association between α‐amylase (green) and CAMKII (magenta) (arrow). Confocal image in (e) shows a primary mouse neuron stained against α‐amylase (green) and glycogen (magenta). The white squares indicate magnification of the area seen in (f and g). Arrows in (f‐g) indicate an close association between α‐amylase and glycogen. Scale bar in (a and e) indicates 5 μm, scale bar in (d and g) indicates 0.5 μm
    Figure Legend Snippet: Cellular localization of alpha (α)‐amylase in primary mouse neurons. Image in (a) shows immunofluorescent staining of α‐amylase with antibody directed against salivary α‐amylase (green) and neuronal marker microtubule‐associated protein 2 (MAP2) (magenta). The α‐amylase immunoreactivity is seen in the cell body (arrowhead) and along the dendrites (arrow). Confocal image in (b) shows a primary mouse neuronal process where α‐amylase staining (green) and phalloidin (magenta) are in close association. Confocal image in (c) shows immunofluorescent staining of primary mouse neuronal process where α‐amylase (green) and synaptotagmin (magenta) are in close association (arrow). Confocal image in (d) shows a close association between α‐amylase (green) and CAMKII (magenta) (arrow). Confocal image in (e) shows a primary mouse neuron stained against α‐amylase (green) and glycogen (magenta). The white squares indicate magnification of the area seen in (f and g). Arrows in (f‐g) indicate an close association between α‐amylase and glycogen. Scale bar in (a and e) indicates 5 μm, scale bar in (d and g) indicates 0.5 μm

    Techniques Used: Staining, Marker

    2) Product Images from "Aberrant Co-localization of Synaptic Proteins Promoted by Alzheimer’s Disease Amyloid-β Peptides: Protective Effect of Human Serum Albumin"

    Article Title: Aberrant Co-localization of Synaptic Proteins Promoted by Alzheimer’s Disease Amyloid-β Peptides: Protective Effect of Human Serum Albumin

    Journal: Journal of Alzheimer's Disease

    doi: 10.3233/JAD-160346

    Effect of amyloid-beta peptides on PSD-95 and synaptotagmin localization. Neurons in primary culture (4 DIV) were incubated for 2 h in Hanks medium in the absence or the presence of 30 μM Aβ 25-35 , Aβ 40 , Aβ 42 , HSA, or HSA-Aβ complexes (C 25–35, C 40, C 42). After incubation, cells were fixed and immunocytochemistry against PSD-95 (in green) and against synaptotagmin (in red) was carried out. Images were taken using confocal microscopy. Orthogonal projections along the z-axis of the images are shown at the bottom and right. Scale bar: 20 μM
    Figure Legend Snippet: Effect of amyloid-beta peptides on PSD-95 and synaptotagmin localization. Neurons in primary culture (4 DIV) were incubated for 2 h in Hanks medium in the absence or the presence of 30 μM Aβ 25-35 , Aβ 40 , Aβ 42 , HSA, or HSA-Aβ complexes (C 25–35, C 40, C 42). After incubation, cells were fixed and immunocytochemistry against PSD-95 (in green) and against synaptotagmin (in red) was carried out. Images were taken using confocal microscopy. Orthogonal projections along the z-axis of the images are shown at the bottom and right. Scale bar: 20 μM

    Techniques Used: Incubation, Immunocytochemistry, Confocal Microscopy

    Quantification of PSD-95, synaptotagmin fluorescence, and co-localization. Fluorescence (Integrated density) was measured in confocal photographs as those depicted in Fig. 8 using NIH Image J Software. Co-localization fluorescence is referred to concurrence points of green (PSD-95) and red (synaptotagmin) fluorescence. Results are expressed as percentages as compared to non-treated cells and are means±SEM ( n ≥24). One-way ANOVA and Dunnett Test were applied in order to compare different treatments vs. control. Distinct characters are used to indicate statistically different groups as compared to the control ( p
    Figure Legend Snippet: Quantification of PSD-95, synaptotagmin fluorescence, and co-localization. Fluorescence (Integrated density) was measured in confocal photographs as those depicted in Fig. 8 using NIH Image J Software. Co-localization fluorescence is referred to concurrence points of green (PSD-95) and red (synaptotagmin) fluorescence. Results are expressed as percentages as compared to non-treated cells and are means±SEM ( n ≥24). One-way ANOVA and Dunnett Test were applied in order to compare different treatments vs. control. Distinct characters are used to indicate statistically different groups as compared to the control ( p

    Techniques Used: Fluorescence, Software

    3) Product Images from "Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting"

    Article Title: Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

    Journal: The EMBO Journal

    doi: 10.15252/embj.201591125

    Snapin-deficient neurons display enlarged presynaptic terminals retaining various degradative organelles A, B Representative electron micrographs (A) and quantitative analysis (B) showing aberrant accumulation of degradative organelles within enlarged presynaptic terminals of snapin − / − cortical neurons at DIV14. Colored arrowheads indicate various types of degradative organelles: red, AVs; blue, endosomal vacuoles; and green, MVBs/LEs. All these organelles were not readily observed at WT presynaptic terminals. C, D Representative images (C) and quantitative analysis (D) showing enhanced intensity of synaptophysin in snapin − / − neurons. Cortical neurons at DIV14 were co-immunostained with antibodies against SV protein synaptophysin (green) and dendritic marker MAP2 (red). E Sequential immunoblots of synaptosomal fractions (Syn), cytosolic fractions (Cytosol), and total brain lysates (Total) showing elevated endolysosomal marker LAMP-1 and autophagy marker LC3-II (red boxes) in synapse-enriched preparations from snapin cKO mice at P40. Snapin is relatively enriched in synaptosomal fractions in WT mouse brains and is almost absent in synaptosomes from snapin cKO animals (green box). Note that synaptic protein levels (i.e., synaptotagmin) are not necessarily increased in synaptosomal preparations from adult snapin cKO mice, while EM and immunocytochemistry revealed increased SVs levels in snapin − / − presynaptic terminals in in vitro culture conditions. A possible explanation for this discrepancy is that oversized and dysfunctional terminals might be eliminated by microglia in adult snapin cKO animals to optimize network activity and survival. Data information: Data were analyzed from the total number of electron micrographs (B) or neurons (D) indicated in parentheses (n) above bar graphs (B) or within bars (D) taken from three pairs of mice for each genotype and expressed as means ± s.e.m. with Student’s t -test (B) and Mann–Whitney U -test (D). P- values: *0.01–0.05; ***0.0001 to 0.001; ****
    Figure Legend Snippet: Snapin-deficient neurons display enlarged presynaptic terminals retaining various degradative organelles A, B Representative electron micrographs (A) and quantitative analysis (B) showing aberrant accumulation of degradative organelles within enlarged presynaptic terminals of snapin − / − cortical neurons at DIV14. Colored arrowheads indicate various types of degradative organelles: red, AVs; blue, endosomal vacuoles; and green, MVBs/LEs. All these organelles were not readily observed at WT presynaptic terminals. C, D Representative images (C) and quantitative analysis (D) showing enhanced intensity of synaptophysin in snapin − / − neurons. Cortical neurons at DIV14 were co-immunostained with antibodies against SV protein synaptophysin (green) and dendritic marker MAP2 (red). E Sequential immunoblots of synaptosomal fractions (Syn), cytosolic fractions (Cytosol), and total brain lysates (Total) showing elevated endolysosomal marker LAMP-1 and autophagy marker LC3-II (red boxes) in synapse-enriched preparations from snapin cKO mice at P40. Snapin is relatively enriched in synaptosomal fractions in WT mouse brains and is almost absent in synaptosomes from snapin cKO animals (green box). Note that synaptic protein levels (i.e., synaptotagmin) are not necessarily increased in synaptosomal preparations from adult snapin cKO mice, while EM and immunocytochemistry revealed increased SVs levels in snapin − / − presynaptic terminals in in vitro culture conditions. A possible explanation for this discrepancy is that oversized and dysfunctional terminals might be eliminated by microglia in adult snapin cKO animals to optimize network activity and survival. Data information: Data were analyzed from the total number of electron micrographs (B) or neurons (D) indicated in parentheses (n) above bar graphs (B) or within bars (D) taken from three pairs of mice for each genotype and expressed as means ± s.e.m. with Student’s t -test (B) and Mann–Whitney U -test (D). P- values: *0.01–0.05; ***0.0001 to 0.001; ****

    Techniques Used: Marker, Western Blot, Mouse Assay, Immunocytochemistry, In Vitro, Activity Assay, MANN-WHITNEY

    4) Product Images from "Selected SALM (Synaptic Adhesion-Like Molecule) Family Proteins Regulate Synapse Formation"

    Article Title: Selected SALM (Synaptic Adhesion-Like Molecule) Family Proteins Regulate Synapse Formation

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4839-09.2010

    SALM3 and SALM5 induce the uptake of synaptotagmin lumenal domain antibodies in contacting axons of cocultured neurons. A–D , HEK293T cells were transfected with SALM2-Ecto, SALM3-Ecto, SALM5-Ecto, or EGFP-f (control), were cocultured with rat hippocampal neurons (DIV 9–12), followed by the incubation of the live neurons with synaptotagmin I (SynTag) lumenal domain antibodies to tag functional presynaptic nerve terminals and double staining for SynTag ( A1 , B1 , C1 , and D1 ) and EGFP ( A2 )/HA (for SALMs-Ecto; B2 , C2 , and D2 ). Scale bar, 15 μm. E , Quantification of the results from A–D . Mean ± SEM (EGFP-f, 5.5 ± 1.0, n = 17; SALM2-Ecto, 3.8 ± 0.1, n = 20; SALM3-Ecto, 25.5 ± 3.1, n = 26; SALM5-Ecto, 25.0 ± 2.6, n = 28; *** p
    Figure Legend Snippet: SALM3 and SALM5 induce the uptake of synaptotagmin lumenal domain antibodies in contacting axons of cocultured neurons. A–D , HEK293T cells were transfected with SALM2-Ecto, SALM3-Ecto, SALM5-Ecto, or EGFP-f (control), were cocultured with rat hippocampal neurons (DIV 9–12), followed by the incubation of the live neurons with synaptotagmin I (SynTag) lumenal domain antibodies to tag functional presynaptic nerve terminals and double staining for SynTag ( A1 , B1 , C1 , and D1 ) and EGFP ( A2 )/HA (for SALMs-Ecto; B2 , C2 , and D2 ). Scale bar, 15 μm. E , Quantification of the results from A–D . Mean ± SEM (EGFP-f, 5.5 ± 1.0, n = 17; SALM2-Ecto, 3.8 ± 0.1, n = 20; SALM3-Ecto, 25.5 ± 3.1, n = 26; SALM5-Ecto, 25.0 ± 2.6, n = 28; *** p

    Techniques Used: Transfection, Incubation, Functional Assay, Double Staining

    5) Product Images from "Regulated delivery of AMPA receptor subunits to the presynaptic membrane"

    Article Title: Regulated delivery of AMPA receptor subunits to the presynaptic membrane

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg059

    Fig. 7. Distribution of AMPA receptor subunits in subcellular fractions from synaptosomes. ( A ) The AMPA receptor subunits GluR2/3 and GluR1, the GluR2/3 binding partner GRIP, the NMDA receptor subunit NR1, the plasma membrane marker Na + /K + ATPase and the synaptic vesicle protein synaptophysin are all clearly detectable in the crude synaptosome fraction (P2). The crude synaptic vesicle fraction (LP2) obtained upon hypo-osmotic lysis of synaptosomes and differential centrifugation, contains GluR2/3, synaptophysin, GluR1 and GRIP. GluR2/3 and, at a much lesser extent, GluR1 and GRIP, are present, together with synaptophysin, in a synaptic vesicle fraction purified by continuous sucrose density gradient (SG-V). ( B ) Western blot analysis of a vesicular fraction immunoisolated from the SG-V fraction, using magnetic beads coated with antibodies directed against synaptotagmin. Note that synaptophysin (syp), synaptobrevin/VAMP2 (syb) and GluR2/3 are co-enriched in the immunoisolated fraction. ( C ) Western blot analysis of a highly purified synaptic vesicle fraction prepared via CPG-V reveals the presence of GluR2 and GluR1, together with synapsin I (syn I), in synaptic vesicles. Note the lack of GRIP immunoreactivity in the purified synaptic vesicles.
    Figure Legend Snippet: Fig. 7. Distribution of AMPA receptor subunits in subcellular fractions from synaptosomes. ( A ) The AMPA receptor subunits GluR2/3 and GluR1, the GluR2/3 binding partner GRIP, the NMDA receptor subunit NR1, the plasma membrane marker Na + /K + ATPase and the synaptic vesicle protein synaptophysin are all clearly detectable in the crude synaptosome fraction (P2). The crude synaptic vesicle fraction (LP2) obtained upon hypo-osmotic lysis of synaptosomes and differential centrifugation, contains GluR2/3, synaptophysin, GluR1 and GRIP. GluR2/3 and, at a much lesser extent, GluR1 and GRIP, are present, together with synaptophysin, in a synaptic vesicle fraction purified by continuous sucrose density gradient (SG-V). ( B ) Western blot analysis of a vesicular fraction immunoisolated from the SG-V fraction, using magnetic beads coated with antibodies directed against synaptotagmin. Note that synaptophysin (syp), synaptobrevin/VAMP2 (syb) and GluR2/3 are co-enriched in the immunoisolated fraction. ( C ) Western blot analysis of a highly purified synaptic vesicle fraction prepared via CPG-V reveals the presence of GluR2 and GluR1, together with synapsin I (syn I), in synaptic vesicles. Note the lack of GRIP immunoreactivity in the purified synaptic vesicles.

    Techniques Used: Binding Assay, Marker, Lysis, Centrifugation, Purification, Western Blot, Magnetic Beads

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    Synaptic Systems synaptotagmin 1 antibody
    Synaptotagmin 1 Antibody, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems synaptotagmin 1 antibody luminal domain
    Manual STED timelapse imaging of synaptic vesicles <t>(synaptotagmin-1_STAR635P).</t> a , STED timelapses of synaptic vesicle clusters in active synapses. b , Distributions of area (left) and aspect ratio (right) for the clusters in calcium-activity-triggered STED timelapses (red) and manual timelapses (black). Box plots show 25–75% IQRs, middle line is mean, and whiskers reach 1.5 times beyond the first and third quartiles. N = 26 clusters in N = 10 cells (red), N = 14 clusters in N = 14 cells (black). Statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: p = 0.90, test statistic = 0.17 (area); p = 0.76, test statistic = 0.20 (aspect ratio). Scale bars, 1 μm.
    Synaptotagmin 1 Antibody Luminal Domain, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synaptotagmin 1 antibody luminal domain/product/Synaptic Systems
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    Synaptic Systems luminal domain of synaptotagmin 1
    Manual STED timelapse imaging of synaptic vesicles <t>(synaptotagmin-1_STAR635P).</t> a , STED timelapses of synaptic vesicle clusters in active synapses. b , Distributions of area (left) and aspect ratio (right) for the clusters in calcium-activity-triggered STED timelapses (red) and manual timelapses (black). Box plots show 25–75% IQRs, middle line is mean, and whiskers reach 1.5 times beyond the first and third quartiles. N = 26 clusters in N = 10 cells (red), N = 14 clusters in N = 14 cells (black). Statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: p = 0.90, test statistic = 0.17 (area); p = 0.76, test statistic = 0.20 (aspect ratio). Scale bars, 1 μm.
    Luminal Domain Of Synaptotagmin 1, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rabbit anti synaptotagmin 11
    Manual STED timelapse imaging of synaptic vesicles <t>(synaptotagmin-1_STAR635P).</t> a , STED timelapses of synaptic vesicle clusters in active synapses. b , Distributions of area (left) and aspect ratio (right) for the clusters in calcium-activity-triggered STED timelapses (red) and manual timelapses (black). Box plots show 25–75% IQRs, middle line is mean, and whiskers reach 1.5 times beyond the first and third quartiles. N = 26 clusters in N = 10 cells (red), N = 14 clusters in N = 14 cells (black). Statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: p = 0.90, test statistic = 0.17 (area); p = 0.76, test statistic = 0.20 (aspect ratio). Scale bars, 1 μm.
    Rabbit Anti Synaptotagmin 11, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Manual STED timelapse imaging of synaptic vesicles (synaptotagmin-1_STAR635P). a , STED timelapses of synaptic vesicle clusters in active synapses. b , Distributions of area (left) and aspect ratio (right) for the clusters in calcium-activity-triggered STED timelapses (red) and manual timelapses (black). Box plots show 25–75% IQRs, middle line is mean, and whiskers reach 1.5 times beyond the first and third quartiles. N = 26 clusters in N = 10 cells (red), N = 14 clusters in N = 14 cells (black). Statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: p = 0.90, test statistic = 0.17 (area); p = 0.76, test statistic = 0.20 (aspect ratio). Scale bars, 1 μm.

    Journal: Nature Methods

    Article Title: Event-triggered STED imaging

    doi: 10.1038/s41592-022-01588-y

    Figure Lengend Snippet: Manual STED timelapse imaging of synaptic vesicles (synaptotagmin-1_STAR635P). a , STED timelapses of synaptic vesicle clusters in active synapses. b , Distributions of area (left) and aspect ratio (right) for the clusters in calcium-activity-triggered STED timelapses (red) and manual timelapses (black). Box plots show 25–75% IQRs, middle line is mean, and whiskers reach 1.5 times beyond the first and third quartiles. N = 26 clusters in N = 10 cells (red), N = 14 clusters in N = 14 cells (black). Statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: p = 0.90, test statistic = 0.17 (area); p = 0.76, test statistic = 0.20 (aspect ratio). Scale bars, 1 μm.

    Article Snippet: For the labeling of active synapses, 1 μl Synaptotagmin-1 antibody luminal domain (1 mg ml−1, Synaptic Systems, 105 3FB) and 1 μl FluoTag-X2 anti-mouse Ig kappa light chain nanobody conjugated to Abberior STAR635P (5 µM, NanoTag Biotechnologies, N1202-Ab635P) were pre-incubated with 98 μl pre-conditioned neuronal medium and incubated at 23 °C for 20 min. Neurons were then incubated with the Synaptotagmin-1 labeling solution for 30 min in a humidified chamber at 37 °C.

    Techniques: Imaging, Activity Assay

    Imaging of synaptic vesicle dynamics with etSTED at 23 Hz. a , (left) Oregon Green 488 BAPTA-1 widefield image. ROI I shows the neurite region in which a calcium event was detected. (right) Single frames of a STED timelapse recording showing synaptotagmin-1_STAR635P-labeled single synaptic vesicles. A line profile drawn across a synaptic vesicle shows a 62.7 nm FWHM. b , Representative 23.3 Hz STED timelapse recording of 30 frames showing the dynamics of single synaptic vesicles in active synapse upon a detected calcium event. In gray, the difference image between the first and second frame. The temporal-color-coded image depicts the mobility of synaptic vesicles in 1260 ms, with the white arrow displaying the directional movement. c , Representative 23.3 Hz STED timelapse recording of 30 frames showing the dynamics of a synaptic vesicle recycling pool in an active synapse upon a detected calcium event. In gray, the difference image between the first and second frame. The temporal-color-coded image depicts the mobility of the synaptic vesicle recycling pool in 1260 ms. N = 379 events, N = 14 cells. Scale bars, 10 μm ( a , widefield), 1 μm ( a widefield inset), 100 nm ( a STED) and 250 nm ( b , c ).

    Journal: Nature Methods

    Article Title: Event-triggered STED imaging

    doi: 10.1038/s41592-022-01588-y

    Figure Lengend Snippet: Imaging of synaptic vesicle dynamics with etSTED at 23 Hz. a , (left) Oregon Green 488 BAPTA-1 widefield image. ROI I shows the neurite region in which a calcium event was detected. (right) Single frames of a STED timelapse recording showing synaptotagmin-1_STAR635P-labeled single synaptic vesicles. A line profile drawn across a synaptic vesicle shows a 62.7 nm FWHM. b , Representative 23.3 Hz STED timelapse recording of 30 frames showing the dynamics of single synaptic vesicles in active synapse upon a detected calcium event. In gray, the difference image between the first and second frame. The temporal-color-coded image depicts the mobility of synaptic vesicles in 1260 ms, with the white arrow displaying the directional movement. c , Representative 23.3 Hz STED timelapse recording of 30 frames showing the dynamics of a synaptic vesicle recycling pool in an active synapse upon a detected calcium event. In gray, the difference image between the first and second frame. The temporal-color-coded image depicts the mobility of the synaptic vesicle recycling pool in 1260 ms. N = 379 events, N = 14 cells. Scale bars, 10 μm ( a , widefield), 1 μm ( a widefield inset), 100 nm ( a STED) and 250 nm ( b , c ).

    Article Snippet: For the labeling of active synapses, 1 μl Synaptotagmin-1 antibody luminal domain (1 mg ml−1, Synaptic Systems, 105 3FB) and 1 μl FluoTag-X2 anti-mouse Ig kappa light chain nanobody conjugated to Abberior STAR635P (5 µM, NanoTag Biotechnologies, N1202-Ab635P) were pre-incubated with 98 μl pre-conditioned neuronal medium and incubated at 23 °C for 20 min. Neurons were then incubated with the Synaptotagmin-1 labeling solution for 30 min in a humidified chamber at 37 °C.

    Techniques: Imaging, Labeling

    Neuronal functional and structural imaging with etSTED. a , Mean Oregon Green 488 BAPTA-1 widefield image from a 10 s timelapse. Boxes indicate detected events (green, true; magenta, false). n = 25 events. b , Extracted calcium curves at detected events (left), manually annotated events (center), and random positions inside the neuron (right). Fluorescence intensity dF/F 0 = (F( t )−F( t 0 ))/F( t 0 ). c , Characterization of true-positive event detection ratio (True) and detected annotated event ratio (Det). n = 8 cells. d , Representative etSTED experiment. e , etSTED performance with the analysis pipeline rapid_signal_spikes. n = 90 events, n = 11 cells. f , Synaptic vesicle dynamics upon calcium signaling. g , Experiment timeline for one widefield frame. h – j , etSTED experiment with calcium signal-triggered STED imaging of synaptotagmin-1_STAR635P. h , Maximum-projected analysis image. Green boxes indicate detected events. i , Zoom-ins of the ratiometric image at the location of two detected events. Green boxes indicate the STED scan area. j , 2.5 Hz etSTED timelapses. White outlines indicate the detected local calcium activity area. Arrows indicate points of structural reorganization. k , Synaptic vesicle cluster analysis. AR, aspect ratio. l , Event detection ratios, compared with the number of true events, for number of events, local or neurite-wide calcium events, and timelapses with synaptotagmin-1-positive vesicles (Syt-1+) and vesicle clusters. n = 186 events, n = 17 cells. m , n , Analysis of synaptic vesicle clusters in calcium-triggered etSTED (red) and manual STED (black) timelapses: MSD with Δ t = 1 frame ( m , left), cluster area ( m , center) and aspect ratio ( m , right), and time-dependent MSD ( n ). m , The statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: P = 0.018, test statistic = 0.48. n , Individual clusters (semi-transparent curves), mean curves (solid lines), and 83% confidence intervals (shaded areas). red: n = 26 clusters, n = 10 cells; black: n = 14 clusters, n = 14 cells. Box plots ( c , m ) show the 25–75% interquartile range, with the middle line representing the mean, and the whiskers reaching 1.5-fold the first and third quartiles. Bar plots ( e , l ) show the mean, and the whiskers reach ±1 s.d. Scale bars, 10 μm ( a , h ), 3 μm ( i ) and 500 nm ( j, k ).

    Journal: Nature Methods

    Article Title: Event-triggered STED imaging

    doi: 10.1038/s41592-022-01588-y

    Figure Lengend Snippet: Neuronal functional and structural imaging with etSTED. a , Mean Oregon Green 488 BAPTA-1 widefield image from a 10 s timelapse. Boxes indicate detected events (green, true; magenta, false). n = 25 events. b , Extracted calcium curves at detected events (left), manually annotated events (center), and random positions inside the neuron (right). Fluorescence intensity dF/F 0 = (F( t )−F( t 0 ))/F( t 0 ). c , Characterization of true-positive event detection ratio (True) and detected annotated event ratio (Det). n = 8 cells. d , Representative etSTED experiment. e , etSTED performance with the analysis pipeline rapid_signal_spikes. n = 90 events, n = 11 cells. f , Synaptic vesicle dynamics upon calcium signaling. g , Experiment timeline for one widefield frame. h – j , etSTED experiment with calcium signal-triggered STED imaging of synaptotagmin-1_STAR635P. h , Maximum-projected analysis image. Green boxes indicate detected events. i , Zoom-ins of the ratiometric image at the location of two detected events. Green boxes indicate the STED scan area. j , 2.5 Hz etSTED timelapses. White outlines indicate the detected local calcium activity area. Arrows indicate points of structural reorganization. k , Synaptic vesicle cluster analysis. AR, aspect ratio. l , Event detection ratios, compared with the number of true events, for number of events, local or neurite-wide calcium events, and timelapses with synaptotagmin-1-positive vesicles (Syt-1+) and vesicle clusters. n = 186 events, n = 17 cells. m , n , Analysis of synaptic vesicle clusters in calcium-triggered etSTED (red) and manual STED (black) timelapses: MSD with Δ t = 1 frame ( m , left), cluster area ( m , center) and aspect ratio ( m , right), and time-dependent MSD ( n ). m , The statistical test used is a two-sample two-sided Kolmogorov–Smirnov test: P = 0.018, test statistic = 0.48. n , Individual clusters (semi-transparent curves), mean curves (solid lines), and 83% confidence intervals (shaded areas). red: n = 26 clusters, n = 10 cells; black: n = 14 clusters, n = 14 cells. Box plots ( c , m ) show the 25–75% interquartile range, with the middle line representing the mean, and the whiskers reaching 1.5-fold the first and third quartiles. Bar plots ( e , l ) show the mean, and the whiskers reach ±1 s.d. Scale bars, 10 μm ( a , h ), 3 μm ( i ) and 500 nm ( j, k ).

    Article Snippet: For the labeling of active synapses, 1 μl Synaptotagmin-1 antibody luminal domain (1 mg ml−1, Synaptic Systems, 105 3FB) and 1 μl FluoTag-X2 anti-mouse Ig kappa light chain nanobody conjugated to Abberior STAR635P (5 µM, NanoTag Biotechnologies, N1202-Ab635P) were pre-incubated with 98 μl pre-conditioned neuronal medium and incubated at 23 °C for 20 min. Neurons were then incubated with the Synaptotagmin-1 labeling solution for 30 min in a humidified chamber at 37 °C.

    Techniques: Functional Assay, Imaging, Fluorescence, Activity Assay

    etSTED experiment in neurons with calcium imaging (Oregon Green 488 BAPTA-1) and STED timelapse imaging of synaptic vesicles (synaptotagmin-1_STAR635P). a , Mean image of all widefield frames with detected events. Boxes are centered on the coordinates of true (green) and false (magenta) detected events. b , Maximum projection of all ratiometric preprocessed widefield frames with detected events. c , Same as b with boxes centered on the coordinates of true (green) and false (magenta) detected events. d , Number of detected events, true events, local events, and neurite-wide events. e , Widefield frame, ratiometric preprocessed frame, zoom-ins of the widefield and ratiometric, and event-triggered STED timelapse (30 frames, 0.99 Hz) of events as numbered and marked in a , c . N = 186 events, N = 17 cells. Boxes marks the center of the detected event. Same scales and time labels apply to all timelapses. Scale bars, 10 μm ( a , b , c , e widefield and ratiometric), 2 μm ( e zooms) and 1 μm ( e STED).

    Journal: Nature Methods

    Article Title: Event-triggered STED imaging

    doi: 10.1038/s41592-022-01588-y

    Figure Lengend Snippet: etSTED experiment in neurons with calcium imaging (Oregon Green 488 BAPTA-1) and STED timelapse imaging of synaptic vesicles (synaptotagmin-1_STAR635P). a , Mean image of all widefield frames with detected events. Boxes are centered on the coordinates of true (green) and false (magenta) detected events. b , Maximum projection of all ratiometric preprocessed widefield frames with detected events. c , Same as b with boxes centered on the coordinates of true (green) and false (magenta) detected events. d , Number of detected events, true events, local events, and neurite-wide events. e , Widefield frame, ratiometric preprocessed frame, zoom-ins of the widefield and ratiometric, and event-triggered STED timelapse (30 frames, 0.99 Hz) of events as numbered and marked in a , c . N = 186 events, N = 17 cells. Boxes marks the center of the detected event. Same scales and time labels apply to all timelapses. Scale bars, 10 μm ( a , b , c , e widefield and ratiometric), 2 μm ( e zooms) and 1 μm ( e STED).

    Article Snippet: For the labeling of active synapses, 1 μl Synaptotagmin-1 antibody luminal domain (1 mg ml−1, Synaptic Systems, 105 3FB) and 1 μl FluoTag-X2 anti-mouse Ig kappa light chain nanobody conjugated to Abberior STAR635P (5 µM, NanoTag Biotechnologies, N1202-Ab635P) were pre-incubated with 98 μl pre-conditioned neuronal medium and incubated at 23 °C for 20 min. Neurons were then incubated with the Synaptotagmin-1 labeling solution for 30 min in a humidified chamber at 37 °C.

    Techniques: Imaging