Journal: Nature Methods

Article Title: Event-triggered STED imaging

doi: 10.1038/s41592-022-01588-y

Figure Lengend Snippet: This shows an etSTED experiment in HeLa cells expressing Dynamin1-EGFP or CD63-pHluorin and using the dynamin_rise or rapid_signal_spikes analysis pipeline. a , Schematic diagram of the dynamin-mediated endocytosis process of interest, with the increase in intensity due to accumulation of dynamin1 at the endocytic site. b , Schematic diagram of the experiment timeline of one widefield frame. I th , intensity threshold; t th , time threshold. c , Example of a triggering widefield frame in an etSTED experiment with HeLa cells expressing Dynamin1-EGFP. d , Two representative events shown in widefield zooms with three frames leading up to the event (right frame). e , Triggered 5.9 Hz 3D STED timelapses of plasma membrane dynamics where cholesterol (cholesterol-Abberior STAR RED) is labeled. n = 53 events, n = 22 cells. f , Schematic diagram of an exocytosis event of interest, with the increase in fluorescence intensity due to unquenching of pHluorin upon pH neutralization of late endosomes (LEs) and multivesicular bodies (MVBs). g , Schematic diagram of the experiment timeline of one widefield frame. h , Example of a triggering widefield image in etSTED experiment with HeLa cells expressing CD63-pHluorin. i , Two representative events shown in widefield zooms (cyan, left) and analysis ratiometric image zooms (gray, right) with the last two frames before the event. j , Triggered 11 Hz 3D STED timelapses of plasma membrane dynamics and accumulation where cholesterol (cholesterol-Abberior STAR RED) is labeled. n = 232 events, N = 29 cells. Asterisks (*) indicate deconvolved frames, and apply to all following frames in the same timelapse. Scale bars, 10 µm ( c , h ), 2 µm ( d , i ), 250 nm ( e , j ).

Article Snippet: To label sphingolipids, primary neuronal cultures were incubated for 1 h at 37 °C and 5% CO 2 with Abberior STAR RED C12 Sphingosyl PE (d17:1/12:20) (5 mg ml −1 in DMSO to a final concentration of 5 μl ml −1 ).

Techniques: Expressing, Clinical Proteomics, Membrane, Labeling, Fluorescence, Neutralization

Journal: Nature Methods

Article Title: Event-triggered STED imaging

doi: 10.1038/s41592-022-01588-y

Figure Lengend Snippet: This shows an etSTED experiment in hippocampal neurons expressing CD63-EGFP and using the vesicle_proximity analysis pipeline. a , Schematic diagram of an endosomal vesicle interaction process, with the increasing proximity due to one labeled vesicle moving towards another stationary vesicle as labeled with CD63-EGFP. b , Schematic diagram of the experiment timeline of one widefield frame. c , Example of a triggering widefield frame. d , Two representative events shown in widefield zooms with three frames leading up to the event (right frame). The green box indicates the area of the STED scan. e , Triggered 2.8 Hz STED timelapses of endosomal vesicle dynamics where sphingosyl PE (sphingosyl-PE_Abberior STAR RED) is labeled. n = 123 events, n = 23 cells. Scale bars, 10 µm ( c ), 2 µm ( d ), 250 nm ( e ). The vesicles involved in the event are marked by symbols (asterisks and arrowhead).

Article Snippet: To label sphingolipids, primary neuronal cultures were incubated for 1 h at 37 °C and 5% CO 2 with Abberior STAR RED C12 Sphingosyl PE (d17:1/12:20) (5 mg ml −1 in DMSO to a final concentration of 5 μl ml −1 ).

Techniques: Expressing, Labeling

Journal: Nature Methods

Article Title: Event-triggered STED imaging

doi: 10.1038/s41592-022-01588-y

Figure Lengend Snippet: a , Widefield images leading up to the triggering frame (last image), showing the increasing proximity of two vesicles (asterisk (mobile) and arrow (stationary)) indicating a potential interaction. b , Representative frames from 2.8 Hz STED timelapses around the detected event, showing the interaction of two vesicles, labeled with either sphingosyl-PE_Abberior STAR RED or cholesterol-Abberior STAR RED. Boxes marks the STED imaged area ( a ). Sphingosyl PE: N = 123 events, N = 22 cells; cholesterol: N = 49 events, N = 8 cells. Same scales and time labels apply to all timelapses. Asterisks (*) indicates deconvolved frames, and applies to all following frames in the same timelapse. Scale bars, 2 μm ( a ), 250 nm ( b ).

Article Snippet: To label sphingolipids, primary neuronal cultures were incubated for 1 h at 37 °C and 5% CO 2 with Abberior STAR RED C12 Sphingosyl PE (d17:1/12:20) (5 mg ml −1 in DMSO to a final concentration of 5 μl ml −1 ).

Techniques: Labeling