FCS in STED Microscopy

Mueller, Veronika; Honigmann, Alf; Ringemann, Christian; Medda, Rebecca; Schwarzmann, Günter; Eggeling, Christian

doi:10.1016/b978-0-12-405539-1.00001-4

Cited by 57 publications

(31 citation statements)

References 88 publications

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“…The general approach for STED-FCS is the same as for traditional FCS. Only it also requires a STED laser, red-shifted in wavelength relative to the fluorescence emission of the specific probe, optical elements for focusing the STED laser to a donut pattern with a zero intensity foci in the middle, and exquisite co-alignment in space and time of the excitation and STED lasers [28, 82]. With this approach it has been possible to tune the observation spot from a diffraction-limited value of around 250 nm and down to ≈15 nm by adjustment of the STED laser power [28, 29].…”

Section: Resultsmentioning

confidence: 99%

“…This corresponds to an accessible time resolution of STED-FCS for focal radii measurements of 40 ⩽ ω x , y ⩽ 250 nm of 60 µ s ⩽ τ D ⩽ 2.3 ms in multi-lamellar lipid layers. The primary error in the STED-FCS data analysis originates from the measurement precision of the lateral radius of the observation spots where typical measurement errors are around 10%, resulting in final errors of the diffusion coefficient of a similar range [28, 29, 82]. The general advantage of this STED-FCS measurement mode is that it mainly relies on comparing relative values (i.e.…”

Section: Resultsmentioning

confidence: 99%

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Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

Lagerholm

Andrade

Clausen

et al. 2017

J. Phys. D: Appl. Phys.

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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 µm2 s−1, in the plasma membrane of live cells at very short length scales, ≈⩽ 100 nm, and time scales, ≈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 ≈0.7–1.0 µm2 s−1, and a compartment size of about 100–150 nm.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

Lagerholm

Andrade

Clausen

et al. 2017

J. Phys. D: Appl. Phys.

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“…To perform VLS-FCS, a calibrated iris diaphragm was added to the parallel path of the 488 nm excitation beam, midway between a parallel beam expander and the objective lens, in order to control the width of the beam. The size of the observation volume was increased by incremental reduction of the iris diameter concentric to the optical axis (12, 4, 2.5, 1.25, 0.9 and 0.5 mm) and by selecting confocal pinholes with incrementally higher diameters (50,75,100,150,200, and 300 µm). This resulted in observation volume radii evenly spaced on a logarithmic scale, w 0 0.3 to 2.5 µm.…”

Section: Vls-fcs Experimentsmentioning

confidence: 99%

“…For fluorophores restricted to two-dimensional diffusion, the observation lengthscale can also be changed by placing the diffusion plane slightly out of focus 70 , or by using the information captured in time-lapsed images [71][72][73] . Recently, the possibility to extend the range of FCS measurements to sub-diffraction observation volumes has been demonstrated by a number of groups, using stimulated emission depletion 44,49,74,75 , zero-mode waveguides 76 or near-field optical probes 77,78 . Beyond the range of accessible observation volume sizes, our study emphasizes the importance of data quality, since it determines the range of lengthscales around w 0 for which the numerical inversion procedure to recover the MSD can be done.…”

Section: On the Importance Of Characterizing Complex Diffusion Procesmentioning

confidence: 99%

Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

et al. 2016

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The diffusion of macromolecules in cells and in complex fluids is often found to deviate from simple Fickian diffusion. One explanation offered for this behavior is that molecular crowding renders diffusion anomalous, where the mean-squared displacement of the particles scales as r 2 ∝ t α with α < 1. Unfortunately, methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) probe diffusion only over a narrow range of lengthscales and cannot directly test the dependence of the mean-squared displacement (MSD) on time. Here we show that variable-lengthscale FCS (VLS-FCS), where the volume of observation is varied over several orders of magnitude, combined with a numerical inversion procedure of the correlation data, allows retrieving the MSD for up to five decades in time, bridging the gap between diffusion experiments performed at different lengthscales. In addition, we show that VLS-FCS provides a way to assess whether the propagator associated with the diffusion is Gaussian or non-Gaussian. We used VLS-FCS to investigate two systems where anomalous diffusion had been previously reported. In the case of dense cross-linked agarose gels, the measured MSD confirmed that the diffusion of small beads was anomalous at short lengthscales, with a cross-over to simple diffusion around ≈ 1 µm, consistent with a caged diffusion process. On the other hand, for solutions crowded with marginally entangled dextran molecules, we uncovered an apparent discrepancy between the MSD, found to be linear, and the propagators at short lengthscales, found to be non-Gaussian. These contradicting features call to mind the "anomalous, yet Brownian" diffusion observed in several biological systems, and the recently proposed "diffusing diffusivity" model.

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“…fluorescence correlation spectroscopy (FCS), stimulated emission depletion (STED) far-field fluorescence nanoscopy, reversible saturable optical (fluorescence) transitions (RESOLFT), reversible saturable optical (fluorescence) transitions (RESOLFT), ground state depletion microscopy followed by individual molecule return (GSDIM), stochastic optical reconstruction microscopy (STORM), or photoactivated localization microscopy (PALM) [2,15,16,17,18,19,20]. Accordingly the generally very high rate for in-plane lateral diffusion of membrane/nano-domain constituents can be accurately tracked by these methods.…”

Section: Introductionmentioning

confidence: 99%

Evidence for the Formation of Symmetric and Asymmetric DLPC-DAPC Lipid Bilayer Domains

Ritter

Schmidt

Jakab

et al. 2013

Cell Physiol Biochem

2,799

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Background/Aims: We investigated if mixtures of the phosphatidylcholine (PC) lipids 1,2-dilauroyl-sn-glycero-3-phosphocholine (C12:0 PC; DLPC) and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (C20:0 PC; DAPC), which differ by eight methylene groups in acyl chain length, lead to the spontaneous formation of distinct lipid rafts and asymmetric bilayers. Methods: The experiments were performed using Atomic Force Microscopy (AFM). Results: We show that DLPC and DAPC mixed at a molar ratio of 1:1 lead to the formation of single, double and triple bilayers with peaks at 6.14 ± 0.11, 13.27 ± 0.17 and 20.54 ± 0.46 nm, respectively (n=750). Within these formations discrete height steps of 0.92 nm can be resolved (n=422). Conclusion: The most frequently observed height steps value of 0.92 nm matches best with the calculated mean lipid hydrophobic thickness difference for asymmetric C12:0 PC and C20:0 PC lipid bilayers of 0.88 nm. This indicates the ability of DLPC and DAPC to form asymmetric lipid bilayers.

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FCS in STED Microscopy

Cited by 57 publications

References 88 publications

Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

Evidence for the Formation of Symmetric and Asymmetric DLPC-DAPC Lipid Bilayer Domains

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