期刊
NANO LETTERS
卷 18, 期 7, 页码 4233-4240出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.nanolett.8b01190
关键词
Diffusion; STED-FCS; scanning FCS; lipids; plasma membrane; simultaneous scanning
类别
资金
- Wolfson Imaging Centre Oxford
- Micron Advanced Bioimaging Unit (Wellcome Trust Strategic Award) [091911]
- EMBO Long term postdoctoral fellowship
- Marie Sldodowska-Curie Intra-European (MEMBRANE DYNAMICS) postdoctoral fellowship
- Wolfson Foundation
- Medical Research Council (MRC) [MC_UU_12010, G0902418, MC_UU_12025]
- MRC/BBSRC/EPSRC [MR/K01577X/1]
- Wellcome Trust [104924/14/Z/14]
- Deutsche Forschungsgemeinschaft (research unit 1905, Structure and function of the peroxisomal translocon)
- Newton-Katip Celebi Institutional Links grant [352333122]
- Oxford internal funds (John Fell Fund and EPA Cephalosporin Fund)
- Marie Curie Career Integration Grant NanodynacTCELLvation [PCIG13-GA-2013-618914]
- BBSRC [BB/P026354/1] Funding Source: UKRI
- MRC [MR/K01577X/1, G0902418, MC_UU_12010/9, MC_UU_00008/9] Funding Source: UKRI
The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED-FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method, line interleaved excitation scanning STED-FCS (LIESS-FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.
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