期刊
JOURNAL OF PHYSICS D-APPLIED PHYSICS
卷 50, 期 6, 页码 -出版社
IOP PUBLISHING LTD
DOI: 10.1088/1361-6463/aa519e
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资金
- Medical Research Council [MC_UU_12009, MC_UU_12010]
- Wolfson Foundation [18272]
- MRC/BBSRC/EPSRC [MR/K015777X/1]
- Wellcome Trust Multi-User Equipment [104924/Z/14/Z]
- Deutsche Forschungsgemeinschaft (DFG) [SFB755, 1905]
- Danish Ministry of Science and Innovation Elite Forsk
- Alfred Benzon Foundation
- Danish Natural Research Foundation
- Danish Ministry of Science
- Wellcome Trust [104924/Z/14/Z] Funding Source: Wellcome Trust
- Medical Research Council [MC_UU_00008/9, MC_UU_12010/9, MR/K01577X/1] Funding Source: researchfish
- MRC [MC_UU_00008/9, MC_UU_12010/9, MR/K01577X/1] Funding Source: UKRI
Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, >= 5 mu m(2) s(-1), in the plasma membrane of live cells at very short length scales, approximate to <= 100 nm, and time scales, approximate to 1-10 ms. This fast diffusion coefficient has been advocated in several high-speed SPT studies, for lipids and membrane proteins alike, but the equivalent has not been detected in STED-FCS measurements. Resolving this ambiguity is important because the assessment of membrane dynamics currently relies heavily on SPT for the determination of heterogeneous diffusion. A possible systematic error in this approach would thus have vast implications in this field. To address this, we have re-visited the analysis procedure for SPT data with an emphasis on the measurement errors and the effect that these errors have on the measurement outputs. We subsequently demonstrate that STED-FCS and SPT data, following careful consideration of the experimental errors of the SPT data, converge to a common interpretation which for the case of a diffusing phospholipid analogue in the plasma membrane of live mouse embryo fibroblasts results in an unhindered, intra-compartment, diffusion coefficient of approximate to 0.7-1.0 mu m(2) s(-1), and a compartment size of about 100-150 nm.
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